Pyrazine-containing compound

ABSTRACT

Compositions and methods related to the amelioration of pancreatitis through the pharmaceutical manipulation of calcium signaling are disclosed. Such compositions and methods may be used to ameliorate symptoms of acute or chronic pancreatitis or to reduce the chance or severity of pancreatitis in an individual at risk of the condition. In other embodiments, disclosed herein are compositions and methods related to the amelioration of viral diseases through the pharmaceutical manipulation of calcium signaling. In further embodiments, disclosed herein are compositions and methods related to the amelioration of Th17-induced diseases through the pharmaceutical manipulation of calcium signaling.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.15/553,531, filed Aug. 24, 2017, which was filed pursuant to 35 U.S.C. §371 as a U.S. National Phase Application of International ApplicationNo. PCT/US2016/019924, filed Feb. 26, 2016, which claims the benefit ofU.S. Provisional Application No. 62/126,386, filed Feb. 27, 2015, eachof which are herein incorporated by reference in their entirety.

BACKGROUND

Acute pancreatitis is the number one cause of gastrointestinalhospitalizations and a major cost burden in the United States. Acutepancreatitis demonstrates a 10-25% mortality rate in severe patients,and a 3-5% mortality rate overall. There is as yet no disease modifyingtherapy available for sufferers of the condition.

SUMMARY OF THE INVENTION

Provided herein are embodiments related to methods of ameliorating thesymptoms of pancreatitis in a mammal such as a person. In otherembodiments, methods of ameliorating the symptoms of a viral infectionin a mammal such as a person are described herein. In furtherembodiments, methods of ameliorating the symptoms of T helper 17 cell(Th17)-induced inflammation and autoimmune diseases are describedherein.

In some embodiments, the methods comprise the steps of identifying aperson in need of amelioration of symptoms of pancreatitis, andadministering an intracellular Calcium signaling inhibitor to saidperson at a dose sufficient to ameliorate said symptoms. In otherembodiments, the methods comprise the steps of identifying a person inneed of amelioration of symptoms of a viral disease, and administeringan intracellular Calcium signaling inhibitor to said person a dosesufficient to ameliorate said symptoms. In further embodiments, themethods comprise the steps of identifying a person in need ofamelioration of symptoms of Th17-induced diseases, and administering anintracellular Calcium signaling inhibitor to said person a dosesufficient to ameliorate said symptoms. In some aspects theintracellular Calcium signaling inhibitor is a SOC channel inhibitor. Insome aspects the intracellular Calcium signaling inhibitor is a CRACchannel inhibitor. In some aspects the intracellular Calcium signalinginhibitor inhibits a channel comprising a STIM1 protein. In some aspectsthe intracellular Calcium signaling inhibitor inhibits a channelcomprising Orai1 protein. In some aspects the intracellular Calciumsignaling inhibits a channel comprising Orai2 protein.

In some aspects the intracellular Calcium signaling inhibitor is acompound having a structure of:

(collectively, “Compound A”), or a pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof. In some embodiments the intracellular Calcium signalinginhibitor is a compound having a structure from the group of Compound Aor a nanoparticle formulation thereof, including a nanoparticlesuspension or emulsion.

In some aspects the intracellular Calcium signaling inhibitor is acompound ofN-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamide.In some aspects the intracellular Calcium signaling inhibitor is acompound ofN-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-vl)pyrazin-2-yl)-2-fluoro-6-methylbenzamideor a pharmaceutically acceptable salt, pharmaceutically acceptablesolvate, or pharmaceutically acceptable prodrug thereof. In some aspectsthe intracellular Calcium signaling inhibitor is chosen from among thecompounds,N-(5-(6-ethoxy-4-methylpyridin-3-yl)pyrazin-2-yl)-2,6-difluorobenzamide,N-(5-(2-ethyl-6-methylbenzo[d]oxazol-5-yl)pyridin-2-yl)-3,5-difluoroisonicotinamide,N-(4-(1-ethyl-3-(thiazol-2-yl)-1H-pyrazol-5-yl)phenyl)-2-fluorobenzamide,N-(5-(1-ethyl-3-(triflouromethyl)-1H-pyrazol-5-yl)pyrazin-2-yl)-2,4,6-trifluorobenzamide,4-chloro-1-methyl-N-(4-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)phenyl)-1H-pyrazole-5-carboxamide,N-(4-(3-(difluoromethyl)-5-methyl-1H-pyrazol-1-yl)-3-fluorophenyl)-2,6-difluorobenzamide,N-(4-(3-(difluoromethyl)-5-methyl-1H-pyrazol-1-yl)-3-fluorophenyl)-2,4,6-trifluorobenzamide,N-(4-(3-(difluoromethyl)-1-methyl-1H-pyrazol-5-yl)-3-fluorophenyl)-2,4,6-trifluorobenzamide,4-chloro-N-(3-fluoro-4-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)phenyl)-1-methyl-1H-pyrazole-5-carboxamide,3-fluoro-4-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)-N-((3-methylisothiazol-4-vl)methyl)aniline,N-(5-(7-chloro-2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-6-yl)pyridin-2-yl)-2,6-difluorobenzamide,N-(2,6-difluorobenzyl)-5-(1-ethyl-3-(thiazol-2-yl)-1H-pyrazol-5-yl)pyrimidin-2-amine,3,5-difluoro-N-(3-fluoro-4-(3-methyl-1-(thiazol-2-yl)-1H-pyrazol-4-yl)phenyl)isonicotinamide,5-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-vl)-N-(2,4,6-trifluorobenzyl)pyridin-2-amine,N-(5-(1-ethyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)pyridin-2-yl)-2,4,6-trifluorobenzamide,N-(5-(5-chloro-2-methylbenzo[d]oxazol-6-yl)pyrazin-2-yl)-2,6-difluorobenzamide,N-(5-(6-ethoxy-4-methylpyridin-3-yl)thiazol-2-yl)-2,3,6-trifluorobenzamide,N-(5-(1-ethyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)pyridin-2-yl)-2,3,6-trifluorobenzamide,2,3,6-trifluoro-N-(3-fluoro-4-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)phenyl)benzamide,2,6-difluoro-N-(4-(5-methyl-2-(trifluoromethyl)oxazol-4-yl)phenyl)benzamide,orN-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamide,(collectively, “Compound A”), or a pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof. In some aspects the symptoms are acute pancreatitissymptoms. In some aspects the symptoms comprise at least one ofinflammation and edema of the pancreas, upper abdominal pain radiatingto the back, left upper quadrant pain radiating to the back, nausea,vomiting, vomiting that is worsened with eating, elevated heart rate,tachycardia, elevated respiratory rate, elevated blood pressure,decreased blood pressure, dehydration, abdominal tenderness, fever,chills, peritonitis, hemodynamic instability, and reflex bowelparalysis. In some aspects the symptoms are severe acute pancreatitissymptoms. In some aspects the symptoms comprise at least one ofpancreatic necrosis and injury to extra-pancreatic organs. In someaspects the symptoms are chronic pancreatitis symptoms. In some aspectsthe symptoms comprise at least one of persistent abdominal pain,digestive defects, malabsorption of fats, pain during food uptake,weight loss, elevation of serum amylase activity, elevation of serumlipase activity, elevation of a CRP inflammatory marker, impairment ofbicarbonate production, elevated fecal elastase levels, elevated serumtrypsinogen levels, pancreatic calcification, elevated serum bilirubinlevels, and elevated alkaline phosphatase levels. In some aspects thesymptoms comprise at least one of elevated ESR levels, elevated IgG4levels, elevated rheumatoid factor, presence of ANA antibody, presenceof antismooth muscle antibody, assay of any of which may indicatechronic pancreatitis in a person In some aspects the symptoms compriseat least one of steatorrhea, Sudan chemical staining of feces or fecalfat excretion of 7 grams or more over a 24 hr period on a 100 g fatdiet, and fecal elastase in a stool sample at a value of less than 200μg/g. In some aspects the symptoms comprise at least one of abdominalpain, increased blood amylase levels, increased blood lipase levels,enlarged pancreas, nausea, vomiting, internal bleeding, bowel paralysis,fever, jaundice, weight loss, and elevated heart rate. In some aspectsthe symptoms comprise elevated serum levels of amylase. In some aspectsthe symptoms comprise elevated serum levels of lipase. In some aspectsthe symptoms comprise findings of necrosis by computed tomography (CT)scan. In some aspects the symptoms comprise premature digestive enzymeactivation. In some aspects the premature digestive enzyme activationoccurs in a pancreas of said person. In some aspects the enzymecomprises trypsin.

Some embodiments relate to methods of preventing or ameliorating asymptom associated with a pancreatic disorder in a person at risk of apancreatic disorder. In some embodiments the method comprises the stepsof identifying a person having a risk factor associated with apancreatic disorder; and administering an intracellular Calciumsignaling inhibitor at a dose sufficient to prevent or ameliorate saidside effect. In some aspects the intracellular Calcium inhibitor is aSOC channel inhibitor. In some aspects the intracellular Calciuminhibitor is a CRAC channel inhibitor. In some aspects the intracellularCalcium signaling inhibitor is a compound having the structure from thegroup of Compound A, or a pharmaceutically acceptable salt,pharmaceutically acceptable solvate, or pharmaceutically acceptableprodrug thereof. In some aspects the intracellular Calcium signalinginhibitor is a compound of,N-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamide.In some aspects the pancreatic disorder comprises an acute pancreatitissymptom. In some aspects the pancreatic disorder comprises a chronicpancreatitis symptom. In some aspects the person has pancreatitis as aresult of being subjected to a drug regimen comprising administration ofat least one of a steroid such as a corticosteroid, prednisolone, an HIVdrug, didanosine, pentamidine, a diuretic, valproic acid,L-asparaginase, azathioprine, estrogen, a statin such as acholesterol-lowering statin, an antihyperglycemic agent, metformin, aglipin such as vildagliptin and sitagliptin, an atypical antipsychotic,clozapine, risperidone, and olanzapine. In some aspects the person isidentified as harboring an inherited form of pancreatitis. In someaspects the person harbors a mutant allele of at least one of trypsin1,encoding trypsinogen, SPINK1, encoding a trypsin inhibitor, and cysticfibrosis transmembrane conductance regulator. In some aspects the personhas pancreatitis as a result of suffering at least one of high bloodcalcium, hypothermia, endoscopic retrograde cholangiopancreatography(ERCP), pancreas divisum, a congenital malformation of the pancreas,diabetes mellitus type 2, pancreatic cancer, pancreatic duct stones,vasculitis, inflammation of the small blood vessels in the pancreas,coxsackievirus infection, and porphyra, such as acute intermittentporphyria and erythropoietic protoporphyria. In some aspects the bodilyhealth condition of said person has been impacted at least one of a gallstone, ethanol poisoning, alcoholism, trauma, mumps, an autoimmunedisorder, a scorpion sting, hyperlipidaemia, hypothermia,hyperparathyroidism, and endoscopic retrograde cholangiopancreatography,azathioprine, and valproic acid. In some aspects the bodily healthcondition of said person has been impacted by at least one of aCoxsackie virus, a Cytomegalovirus, a Hepatitis B virus, a Herpessimplex virus, Mumps, a Varicella-zoster virus, a Legionella bacterium,a Leptospira bacterium, a Mycoplasma bacterium, a Salmonella bacterium,an Aspergillus fungus, an Ascaris parasite, a Cryptosporidium cell and aToxoplasma cell.

Some embodiments relate to methods of preventing or ameliorating asymptom associated with a viral disease in a person at risk of a viraldisease. In some embodiments the method comprises the steps of:identifying a person having a risk factor associated with a viraldisease; and administering an intracellular Calcium signaling inhibitorat a dose sufficient to prevent or ameliorate said side effect. Someembodiments relate to a composition for use of ameliorating the symptomsof viral disease in a person comprising the steps of identifying aperson in need of amelioration of symptoms of viral disease, andadministering an intracellular Calcium signaling inhibitor to saidperson at a dose sufficient to ameliorate said symptoms. In some aspectsthe intracellular Calcium signaling inhibitor is a CRAC channelinhibitor. In some aspects the intracellular Calcium signaling inhibitorinhibits a channel comprising a STIM1 protein. In some aspects theintracellular Calcium signaling inhibitor inhibits a channel comprisingOrai1 protein. In some aspects the intracellular Calcium signalinginhibits a channel comprising Orai2 protein. In some aspects theintracellular Calcium signaling inhibitor is a compound having thestructure from the group of Compound A, or a pharmaceutically acceptablesalt, pharmaceutically acceptable solvate, or pharmaceuticallyacceptable prodrug thereof. In some aspects the intracellular Calciumsignaling inhibitor is a compound of,N-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamide.In some aspects the intracellular Calcium signaling inhibitor is acompound ofN-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamideor a pharmaceutically acceptable salt, pharmaceutically acceptablesolvate, or pharmaceutically acceptable prodrug thereof. In some aspectsthe intracellular Calcium signaling inhibitor blocks budding of theviral disease. In some aspects, the viral disease is a hemorrhagic fevervirus. In further aspects, the hemorrhagic fever virus is an arenavirus,a filovirus, a bunyavirus, a flavivirus, a rhabdovirus, or combinationsthereof. In even further aspects, the hemorrhagic fever virus is Ebolavirus, Marburg virus, Lassa virus, Junin virus, Rotavirus, West Nilevirus, Zika virus, Coxsackievirus, Hepatitis B virus, Epstein Barrvirus, dengue virus, or Rift Valley virus. In some aspects, the symptomsare fever or bleeding diathesis. In further aspects, the symptoms of aviral disease are at least one of flushing of the face, flushing of thechest, petechiae, capillary leak, bleeding, swelling, edema,hypotension, shock, malaise, muscle pain, headache, vomiting, diarrhea,or combinations thereof.

Some embodiments relate to methods of preventing or ameliorating asymptom associated with a Th17-induced disease in a person at risk of aTh17-induced disease. In some embodiments the method comprises the stepsof: identifying a person having a risk factor associated with aTh17-induced disease; and administering an intracellular Calciumsignaling inhibitor at a dose sufficient to prevent or ameliorate saidside effect. Some embodiments relate to a composition for use ofameliorating the symptoms of Th17-induced disease in a person comprisingthe steps of identifying a person in need of amelioration of symptoms ofpancreatitis, and administering an intracellular Calcium signalinginhibitor to said person at a dose sufficient to ameliorate saidsymptoms. In some aspects the intracellular Calcium signaling inhibitoris a CRAC channel inhibitor. In some aspects the intracellular Calciumsignaling inhibitor inhibits a channel comprising a STIM1 protein. Insome aspects the intracellular Calcium signaling inhibitor inhibits achannel comprising Orai1 protein. In some aspects the intracellularCalcium signaling inhibits a channel comprising Orai2 protein. In someaspects the intracellular Calcium signaling inhibitor is a compoundhaving the structure from the group of Compound A, or a pharmaceuticallyacceptable salt, pharmaceutically acceptable solvate, orpharmaceutically acceptable prodrug thereof. In some aspects theintracellular Calcium signaling inhibitor is a compound of,N-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamide.In some aspects the intracellular Calcium signaling inhibitor is acompound ofN-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamideor a pharmaceutically acceptable salt, pharmaceutically acceptablesolvate, or pharmaceutically acceptable prodrug thereof. In some aspectsthe intracellular Calcium signaling inhibitor blocks differentiation ofTh17 cells. In some aspects the Th17-induced disease is a chronicinflammatory disease. In further aspects, the chronic inflammatorydisease is hay fever, periodontitis, atherosclerosis, rheumatoidarthritis, or cancer. In other aspects the Th17-induced disease is anautoimmune disease. In further aspects, the autoimmune disease isrheumatoid arthritis, lupus, celiac disease, psoriasis, Sjorgen'ssyndrome, polymyalgia rheumatica, multiple sclerosis, ankylosingspondylitis, type 1 diabetes, alopecia areata, vasculitis, or temporalarteritis. In some aspects, the symptoms of Th17-induced diseases are atleast one of localized reddening, swelling, heat, pain, stiffness,fever, chills, fatigue, headache, or appetite loss. In some aspects, thesymptoms occur on the body of the person comprising the torso, arms,hands, fingers, legs, feet, toes, head, neck, bones, joints, throat,sinuses, eyes, or combinations thereof.

Some embodiments relate to a composition comprising an intracellularCalcium signaling inhibitor and at least one drug associated with anegative impact on pancreatic activity. In some aspects the drug isselected from the list consisting of: a steroid such as acorticosteroid, prednisolone, an HIV drug, didanosine, pentamidine, adiuretic, valproic acid, L-asparaginase, azathioprine, estrogen, astatin such as a cholesterol-lowering statin, an antihyperglycemicagent, metformin, a glipin such as vildagliptin and sitagliptin, anatypical antipsychotic, clozapine, risperidone, and olanzapine,azathioprine, and valproic acid. In some aspects the intracellularCalcium signaling inhibitor is an SOC inhibitor. In some aspects theintracellular Calcium signaling inhibitor is a CRAC inhibitor. In someaspects the intracellular Calcium signaling inhibitor is a compoundhaving the structure from the group of Compound A, or a pharmaceuticallyacceptable salt, pharmaceutically acceptable solvate, orpharmaceutically acceptable prodrug thereof. In some aspects theintracellular Calcium signaling inhibitor is a compound of,N-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamide.In some aspects the intracellular Calcium signaling inhibitor is acompound ofN-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamideor a pharmaceutically acceptable salt, pharmaceutically acceptablesolvate, or pharmaceutically acceptable prodrug thereof.

Some aspects relate to a dosing regimen comprising administration to anindividual of a drug associated with a negative impact on pancreaticactivity, and administration of an intracellular Calcium signalinginhibitor. In some aspects the drug is selected from the list consistingof: a steroid such as a corticosteroid, prednisolone, an HIV drug,didanosine, pentamidine, a diuretic, valproic acid, L-asparaginase,azathioprine, estrogen, a statin such as a cholesterol-lowering statin,an antihyperglycemic agent, metformin, a glipin such as vildagliptin andsitagliptin, an atypical antipsychotic, clozapine, risperidone, andolanzapine, azathioprine, and valproic acid. In some aspects theintracellular Calcium signaling inhibitor is an SOC inhibitor. In someaspects the intracellular Calcium signaling inhibitor is a CRACinhibitor. In some aspects the intracellular Calcium signaling inhibitoris a compound having the structure from the group of Compound A, or apharmaceutically acceptable salt, pharmaceutically acceptable solvate,or pharmaceutically acceptable prodrug thereof. In some aspects theintracellular Calcium signaling inhibitor is a compound of,N-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamide.In some aspects the intracellular Calcium signaling inhibitor is acompound ofN-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamideor a pharmaceutically acceptable salt, pharmaceutically acceptablesolvate, or pharmaceutically acceptable prodrug thereof.

Some embodiments relate to a composition for use of ameliorating thesymptoms of pancreatitis in a person comprising the steps of identifyinga person in need of amelioration of symptoms of pancreatitis, andadministering an intracellular Calcium signaling inhibitor to saidperson at a dose sufficient to ameliorate said symptoms. In some aspectsthe intracellular Calcium signaling inhibitor is a SOC channelinhibitor. In some aspects the intracellular Calcium signaling inhibitoris a CRAC channel inhibitor. In some aspects the intracellular Calciumsignaling inhibitor inhibits a channel comprising a STIM1 protein. Insome aspects the intracellular Calcium signaling inhibitor inhibits achannel comprising Orai1 protein. In some aspects the intracellularCalcium signaling inhibits a channel comprising Orai2 protein. In someaspects the intracellular Calcium signaling inhibitor is a compoundhaving the structure from the group of Compound A, or a pharmaceuticallyacceptable salt, pharmaceutically acceptable solvate, orpharmaceutically acceptable prodrug thereof. In some aspects theintracellular Calcium signaling inhibitor is a compound of,N-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamide.In some aspects the intracellular Calcium signaling inhibitor is acompound ofN-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamideor a pharmaceutically acceptable salt, pharmaceutically acceptablesolvate, or pharmaceutically acceptable prodrug thereof. In some aspectsthe composition further comprises a painkiller medication. In someaspects the painkiller medication comprises an opiate. In some aspectsthe painkiller medication comprises morphine. In some aspects thesymptoms are acute pancreatitis symptoms. In some aspects the symptomscomprise at least one of inflammation and edema of the pancreas, upperabdominal pain radiating to the back, left upper quadrant pain radiatingto the back, nausea, vomiting, vomiting that is worsened with eating,elevated heart rate, tachycardia, elevated respiratory rate, elevatedblood pressure, decreased blood pressure, dehydration, abdominaltenderness, fever, chills, peritonitis, hemodynamic instability, andreflex bowel paralysis. In some aspects the symptoms are severepancreatitis symptoms. In some aspects the symptoms comprise at leastone of pancreatic necrosis and injury to extra-pancreatic organs. Insome aspects the symptoms are chronic pancreatitis symptoms. In someaspects the symptoms comprise at least one of persistent abdominal pain,digestive defects, malabsorption of fats, pain during food uptake,weight loss, elevation of serum amylase activity, elevation of serumlipase activity, elevation of a CRP inflammatory marker, impairment ofbicarbonate production, elevated fecal elastase levels, elevated serumtrypsinogen levels, pancreatic calcification, elevated serum bilirubinlevels, and elevated alkaline phosphatase levels. In some aspects thesymptoms comprise at least one of elevated ESR levels, elevated IgG4levels, elevated rheumatoid factor, presence of ANA antibody, presenceof antismooth muscle antibody, assay of any of which may indicatechronic pancreatitis in a person. In some aspects the symptoms compriseat least one of steatorrhea, Sudan chemical staining of feces or fecalfat excretion of 7 grams or more over a 24 hr period on a 100 g fatdiet, and fecal elastase in a stool sample at a value of less than 200μg/g. In some aspects the symptoms comprise at least one of abdominalpain, increased blood amylase levels, increased blood lipase levels,enlarged pancreas, nausea, vomiting, internal bleeding, bowel paralysis,fever, jaundice, weight loss, and elevated heart rate. In some aspectsthe symptoms comprise premature digestive enzyme activation. In someaspects the premature digestive enzyme activation occurs in a pancreasof said person. In some aspects the enzyme comprises trypsin.

Some aspects relate to a composition for use of preventing orameliorating a symptom associated with a pancreatic disorder in a personat risk of a pancreatic disorder, comprising the steps of: identifying aperson having a risk factor associated with a pancreatic disorder; andadministering an intracellular Calcium signaling inhibitor at a dosesufficient to prevent or ameliorate said side effect. In some aspectsthe intracellular Calcium inhibitor is a SOC channel inhibitor. In someaspects the intracellular Calcium inhibitor is a CRAC channel inhibitor.In some aspects the intracellular Calcium signaling inhibitor is acompound having the structure from the group of Compound A, or apharmaceutically acceptable salt, pharmaceutically acceptable solvate,or pharmaceutically acceptable prodrug thereof. In some aspects theintracellular Calcium signaling inhibitor is a compound of,N-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamide.In some aspects the intracellular Calcium signaling inhibitor is acompound ofN-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamideor a pharmaceutically acceptable salt, pharmaceutically acceptablesolvate, or pharmaceutically acceptable prodrug thereof. In some aspectsthe pancreatic disorder comprises an acute pancreatitis symptom. In someaspects the pancreatic disorder comprises a chronic pancreatitissymptom. In some aspects the person is subjected to a drug regimencomprising administration of at least one of a steroid such as acorticosteroid, prednisolone, an HIV drug, didanosine, pentamidine, adiuretic, valproic acid, L-asparaginase, azathioprine, estrogen, astatin such as a cholesterol-lowering statin, an antihyperglycemicagent, metformin, a glipin such as vildagliptin and sitagliptin, anatypical antipsychotic, clozapine, risperidone, and olanzapine. In someaspects the person is identified as harboring an inherited form ofpancreatitis. In some aspects the person harbors a mutant allele of atleast one of trypsin, encoding trypsinogen, SPINK1, encoding a trypsininhibitor, and cystic fibrosis transmembrane conductance regulator. Insome aspects the person has suffered at least one of high blood calcium,hypothermia, endoscopic retrograde cholangiopancreatography (ERCP),pancreas divisum, a congenital malformation of the pancreas, diabetesmellitus type 2, pancreatic cancer, pancreatic duct stones, vasculitis,inflammation of the small blood vessels in the pancreas, coxsackie virusinfection, and porphyra, such as acute intermittent porphyria anderythropoietic protoporphyria. In some aspects the bodily healthcondition of said person has been impacted at least one of a gall stone,ethanol poisoning, alcoholism, trauma, mumps, an autoimmune disorder, ascorpion sting, hyperlipidaemia, hypothermia, hyperparathyroidism, andendoscopic retrograde cholangiopancreatography, azathioprine, andvalproic acid. In some aspects the bodily health condition of saidperson has been impacted by at least one of a Cytomegalovirus, aHepatitis B virus, a Herpes simplex virus, Mumps, a Varicella-zostervirus, a Legionella bacterium, a Leptospira bacterium, a Mycoplasmabacterium, a Salmonella bacterium, an Aspergillus fungus, an Ascarisparasite, a Cryptosporidium cell and a Toxoplasma cell.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

In particular, PCT Publication No. WO 2011/139489 A2, published Nov. 10,2011, is hereby incorporated by reference in its entirety; PCTPublication No. WO 2009/035818, published Mar. 19, 2009, is herebyincorporated by reference in its entirety; PCT Publication No. WO2010/025295, published Jun. 17, 2010, is hereby incorporated byreference in its entirety; PCT Publication No. WO 2010/027875, publishedJun. 10, 2010, is hereby incorporated by reference in its entirety; PCTPublication No. WO 2011/034962, published Jul. 28, 2011, is herebyincorporated by reference in its entirety; PCT Publication No. WO2011/139489, published Jan. 26, 2012, is hereby incorporated byreference in its entirety; PCT Publication No. WO 2011/139765 publishedMar. 8, 2012, is hereby incorporated by reference in its entirety; PCTPublication No. WO 2012/027710, published May 18, 2012, is herebyincorporated by reference in its entirety; PCT Publication No. WO2012/170931, published Feb. 21, 2013 is hereby incorporated by referencein its entirety; PCT Publication No. WO 2012/170951, published Apr. 25,2013, is hereby incorporated by reference in its entirety; PCTPublication No. WO 2013/059666, published Apr. 25, 2013, is herebyincorporated by reference in its entirety; PCT Publication No. WO2013/059677, published Apr. 25, 2013, is hereby incorporated byreference in its entirety; PCT Publication No. WO 2014/043715, publishedMar. 20, 2014, is hereby incorporated by reference in its entirety; andPCT Publication No. WO 2014/059333, published Apr. 17, 2014, is herebyincorporated by reference in its entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A shows mouse Acinar cell necrosis upon treatment with TLCS.

FIG. 1B shows human Acinar cell necrosis upon treatment with TLCS.

FIG. 2A shows histopathology scores for Caelurin-induced acutepancreatitits.

FIG. 2B shows histopathology scores for TLCS-induced acutepancreatitits.

FIG. 2C shows histopathology scores for FAEE-induced acutepancreatitits.

FIG. 3A shows an IC₅₀ determination forN-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamide(“Compound I”).

FIG. 3B shows an IC₅₀ determination for2,6-Difluoro-N-(1-(4-hydroxy-2-(trifluoromethyl)benzyl)-1H-pyrazol-3-yl)benzamide(“GSK-7975A”).

FIG. 4A shows Calcium uptake in the absence of a CRAC inhibitor.

FIG. 4B shows Calcium uptake in the presence of a CRAC inhibitor.

FIG. 5A shows Calcium entry in the presence of CRAC inhibitor CompoundI.

FIG. 5B shows Calcium entry in the presence of CRAC inhibitor GSK-7975A.

FIG. 6A shows Calcium entry in the absence of a CRAC inhibitor.

FIG. 6B shows relative Calcium entry in the presence of CRAC inhibitorsCompound I and GSK-7975A compared to a control.

FIG. 7 shows IC₅₀ values for Compound I against a number of cytokines.

FIG. 8A shows histopathology scores for a range of concentrations ofCompound I.

FIG. 8B shows Compound I concentrations in the pancreas as a function ofdose.

FIG. 9A shows serum Amylase levels in uninduced (normal) mice and micefor which acute pancreatitis is induced, in the presence and absence ofa CRAC inhibitor.

FIG. 9B shows serum Lipase levels in uninduced (normal) mice and micefor which acute pancreatitis is induced, in the presence and absence ofa CRAC inhibitor.

FIG. 10 shows histopathology scores for therapeutically andprophylactically treated mice upon acute pancreatitis induction.

FIG. 11 shows TLCS-induced Calcium levels.

FIG. 12 shows mouse Acinar cell Amylase release levels.

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions disclosed herein are used for modulatingintracellular calcium to ameliorate or prevent symptoms of pancreatitis.In some aspects, the pancreatitis is acute pancreatitis. In some aspectsthe pancreatitis is chronic pancreatitis. In some embodiments, methodsand compositions disclosed herein are used for modulating intracellularcalcium to ameliorate or prevent symptoms of a viral disease. In someaspects the viral disease is a hemorrhagic fever virus. In some aspects,the hemorrhagic fever virus is an arenavirus, a filovirus, a bunyavirus,a flavivirus, a rhabdovirus, or combinations thereof. Hemorrhagic feverviruses include, by way of non-limiting examples, Ebola virus, Marburgvirus, Lassa virus, Junin virus, Rotavirus. West Nile virus, Zika virus,Coxsackievirus. Hepatitis B virus, Epstein Barr virus, etc. In furtherembodiments, methods and compositions disclosed herein are used formodulating intracellular calcium to ameliorate or prevent symptoms ofTh17-induced diseases. In some aspects, the Th17-induced disease is aninflammatory disease. In further aspects, the Th17-induced disease is anautoimmune disorder. In some aspects, compounds provided herein modulateSOC channel activity. In some aspects, methods and compounds providedherein modulate CRAC channel activity. In another aspect, compoundsprovided herein modulate STIM protein activity. In another aspect,methods and compounds provided herein modulate Orai protein activity. Inanother aspect, methods and compounds provided herein modulate thefunctional interactions of STIM proteins with Orai proteins. In anotheraspect, methods and compounds provided herein reduce the number offunctional SOC channels. In another aspect, methods and compoundsprovided herein reduce the number of functional CRAC channels. In someaspects, methods and compounds described herein are SOC channelblockers. In some aspects, methods and compounds described herein areCRAC channel blockers or CRAC channel modulators.

Calcium plays a vital role in cell function and survival. For example,calcium is a key element in the transduction of signals into and withincells. Cellular responses to growth factors, neurotransmitters, hormonesand a variety of other signal molecules are initiated throughcalcium-dependent processes.

Virtually all cell types depend in some manner upon the generation ofcytoplasmic Ca²⁺ signals to regulate cell function, or to triggerspecific responses. Cytosolic Ca²⁺ signals control a wide array ofcellular functions ranging from short-term responses such as contractionand secretion to longer-term regulation of cell growth andproliferation. Usually, these signals involve some combination ofrelease of Ca²⁺ from intracellular stores, such as the endoplasmicreticulum (ER), and influx of Ca²⁺ across the plasma membrane. In oneexample, cell activation begins with an agonist binding to a surfacemembrane receptor, which is coupled to phospholipase C (PLC) through aG-protein mechanism. PLC activation leads to the production of inositol1,4,5-triphosphate (IP₃), which in turn activates the IP₃ receptorcausing release of Ca²⁺ from the ER. The fall in ER Ca²⁺ then signals toactivate plasma membrane store-operated calcium (SOC) channels.

Store-operated calcium (SOC) influx is a process in cellular physiologythat controls such diverse functions such as, but not limited to,refilling of intracellular Ca²⁺ stores (Putney et al. Cell, 75, 199-201,1993), activation of enzymatic activity (Fagan et al., J. Biol. Chem.275:26530-26537, 2000), gene transcription (Lewis, Annu. Rev. Immunol.19:497-521, 2001), cell proliferation (Nunez et al., J. Physiol. 571.1,57-73, 2006), and release of cytokines (Winslow et al., Cur. Opin.Immunol. 15:299-307, 2003). In some nonexcitable cells, e.g., bloodcells, immune cells, hematopoietic cells, T lymphocytes and mast cells,pancreatic acinar cells (PACs), epithelial and ductal cells of otherglands (eg salivary glands), endothelial and endothelial progenitorcells, SOC influx occurs through calcium release-activated calcium(CRAC) channels, a type of SOC channel.

The calcium influx mechanism has been referred to as store-operatedcalcium entry (SOCE). Stromal interaction molecule (STIM) proteins arean essential component of SOC channel function, serving as the sensorsfor detecting the depletion of calcium from intracellular stores and foractivating SOC channels.

Calcium Homeostasis

Cellular calcium homeostasis is a result of the summation of regulatorysystems involved in the control of intracellular calcium levels andmovements. Cellular calcium homeostasis is achieved, at least in part,by calcium binding and by movement of calcium into and out of the cellacross the plasma membrane and within the cell by movement of calciumacross membranes of intracellular organelles including, for example, theendoplasmic reticulum, sarcoplasmic reticulum, mitochondria andendocytic organelles including endosomes and lysosomes.

Movement of calcium across cellular membranes is carried out byspecialized proteins. For example, calcium from the extracellular spacecan enter the cell through various calcium channels and a sodium/calciumexchanger and is actively extruded from the cell by calcium pumps andsodium/calcium exchangers. Calcium can also be released from internalstores through inositol trisphosphate or ryanodine receptors and can betaken up by these organelles by means of calcium pumps.

Calcium can enter cells by any of several general classes of channels,including but not limited to, voltage-operated calcium (VOC) channels,store-operated calcium (SOC) channels, and sodium/calcium exchangersoperating in reverse mode. VOC channels are activated by membranedepolarization and are found in excitable cells like nerve and muscleand are for the most part not found in nonexcitable cells. Under someconditions, Ca²⁺ can enter cells via Na+-Ca²⁺ exchangers operating inreverse mode.

Endocytosis provides another process by which cells can take up calciumfrom the extracellular medium through endosomes. In addition, somecells, e.g., exocrine cells, can release calcium via exocytosis.

Cytosolic calcium concentration is tightly regulated with resting levelsusually estimated at approximately 0.1 μM in mammalian cells, whereasthe extracellular calcium concentration is typically about 2 mM. Thistight regulation facilitates transduction of signals into and withincells through transient calcium flux across the plasma membrane andmembranes of intracellular organelles. There is a multiplicity ofintracellular calcium transport and buffer systems in cells that serveto shape intracellular calcium signals and maintain the low restingcytoplasmic calcium concentration. In cells at rest, the principalcomponents involved in maintaining basal calcium levels are calciumpumps and leak pathways in both the endoplasmic reticulum and plasmamembrane. Disturbance of resting cytosolic calcium levels can affecttransmission of calcium-dependent signals and give rise to defects in anumber of cellular processes. For example, cell proliferation involves aprolonged calcium signaling sequence. Other cellular processes thatinvolve calcium signaling include, but are not limited to, secretion,transcription factor signaling, and fertilization.

Cell-surface receptors that activate phospholipase C (PLC) createcytosolic Ca²⁺ signals from intra- and extra-cellular sources. Aninitial transient rise of [Ca²⁺]i (intracellular calcium concentration)results from the release of Ca²⁺ from the endoplasmic reticulum (ER),which is triggered by the PLC product, inositol-1,4,5-trisphosphate(IP₃), opening IP₃ receptors in the ER (Streb et al. Nature, 306, 67-69,1983). A subsequent phase of sustained Ca²⁺ entry across the plasmamembrane then ensues, through specialized store operated calcium (SOC)channels (in the case of non-excitable cells like immune PAC cells, theSOC channels are calcium release-activated calcium (CRAC) channels) inthe plasma membrane. Store-operated Ca2+ entry (SOCE) is the process inwhich the emptying of Ca²⁺ stores itself activates Ca²⁺ channels in theplasma membrane to help refill the stores (Putney, Cell Calcium, 7,1-12, 1986; Parekh et al., Physiol. Rev. 757-810; 2005). SOCE does morethan simply provide Ca2+ for refilling stores, but can itself generatesustained Ca²⁺ signals that control such essential functions as geneexpression, cell metabolism and exocytosis (Parekh and Putney, Physiol.Rev. 85, 757-810 (2005).

In lymphocytes and mast cells, activation of antigen or Fc receptors,respectively causes the release of Ca²⁺ from intracellular stores, whichin turn leads to Ca²⁺ influx through CRAC channels in the plasmamembrane. The subsequent rise in intracellular Ca²⁺ activatescalcineurin, a phosphatase that regulates the transcription factor NFAT.In resting cells, NFAT is phosphorylated and resides in the cytoplasm,but when dephosphorylated by calcineurin, NFAT translocates to thenucleus and activates different genetic programs depending onstimulation conditions and cell type. In response to infections andduring transplant rejection, NFAT partners with the transcription factorAP-1 (Fos-Jun) in the nucleus of “effector” T cells, therebytrans-activating cytokine genes, genes that regulate T cellproliferation and other genes that orchestrate an active immune response(Rao et al., Annu Rev Immunol., 1997:15:707-47). In contrast, in T cellsrecognizing self-antigens, NFAT is activated in the absence of AP-1, andactivates a transcriptional program known as “anergy” that suppressesautoimmune responses (Macian et al., Transcriptional mechanismsunderlying lymphocyte tolerance. Cell, 2002 Jun. 14; 109(6):719-31). Ina subclass of T cells known as regulatory T cells which suppressautoimmunity mediated by self-reactive effector T cells, NFAT partnerswith the transcription factor FOXP3 to activate genes responsible forsuppressor function (Wu et al., Cell, 2006 Jul. 28:126(2):375-87;Rudensky A Y, Gavin M. Zheng Y. Cell, 2006 Jul. 28:126(2):253-256).

The endoplasmic reticulum (ER) carries out a variety processes. The ERhas a role as both a Ca²⁺ sink and an agonist-sensitive Ca²⁺ store, andprotein folding/processing takes place within its lumen. In the lattercase, numerous Ca²⁺-dependent chaperone proteins ensure that newlysynthesized proteins are folded correctly and sent off to theirappropriate destination. The ER is also involved in vesicle trafficking,release of stress signals, regulation of cholesterol metabolism, andapoptosis. Many of these processes require intraluminal Ca²⁺ and proteinmisfolding, ER stress responses, and apoptosis can all be induced bydepleting the ER of Ca²⁺ for prolonged periods of time. Because itcontains a finite amount of Ca²⁺, it is clear that ER Ca²⁺ content mustfall after release of that Ca²⁺ during stimulation. However, to preservethe functional integrity of the ER, it is vital that the Ca²⁺ contentdoes not fall too low or is maintained at least at a low level.Replenishment of the ER with Ca²⁺ is therefore a central process to alleukaryotic cells. Because a fall in ER Ca²⁺ content activatesstore-operated Ca²⁺ channels in the plasma membrane, a major function ofthis Ca²⁺ entry pathway is believed to be maintenance of ER Ca²⁺ levelsthat are necessary for proper protein synthesis and folding. However,store-operated Ca²⁺ channels have other important roles.

The understanding of store-operated calcium entry was provided byelectrophysiological studies which established that the process ofemptying the stores activated a Ca²⁺ current in mast cells called Ca²⁺release-activated Ca²⁺ current or ICRAC. ICRAC is non-voltage activated,inwardly rectifying, and remarkably selective for Ca²⁺. It is found inseveral cell types mainly of hemapoietic origin. ICRAC is not the onlystore-operated current, and it is now apparent that store-operatedinflux encompasses a family of Ca²⁺-permeable channels, with differentproperties in different cell types. ICRAC was the first store-operatedCa²⁺ current to be described and remains a popular model for studyingstore-operated influx.

Store-operated calcium channels can be activated by any procedure thatempties ER Ca²⁺ stores; it does not seem to matter how the stores areemptied, the net effect is activation of store-operated Ca²⁺ entry.Physiologically, store emptying is evoked by an increase in the levelsof IP₃ or other Ca²⁺-releasing signals followed by Ca²⁺ release from thestores. But there are several other methods for emptying stores. Thesemethods include the following:

-   -   1) elevation of IP; in the cytosol (following receptor        stimulation or, dialyzing the cytosol with IP₃ itself or related        congeners like the nonmetabolizable analog Ins(2,4,5)P₃);    -   2) application of a Ca²⁺ ionophore (e.g., ionomycin) to        permeabilize the ER membrane;    -   3) dialyzing the cytoplasm with high concentrations of Ca²⁺        chelators (e.g., EGTA or BAPTA), which chelate Ca²⁺ that leaks        from the stores and hence prevent store refilling;    -   4) exposure to the sarcoplasmic/endoplasmic reticulum        Ca²⁺-ATPase (SERCA) inhibitors like thapsigargin, cyclopiazonic        acid, and di-tert-butylhydroquinone;    -   5) sensitizing the IP; receptors to resting levels of InsP3 with        agents like thimerosal; and    -   6) loading membrane-permeable metal Ca²⁺ chelators like        N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylene diamine (TPEN)        directly into the stores.        Through mass action, TPEN lowers free intraluminal Ca²⁺        concentration without changing total store Ca²⁺ such that the        store depletion-dependent signal is generated.

These methods of emptying stores are not devoid of potential problems.The key feature of store-operated Ca²⁺ entry is that it is the fall inCa²⁺ content within the stores and not the subsequent rise incytoplasmic Ca²⁺ concentration that activates the channels. However,ionomycin and SERCA pump blockers generally cause a rise in cytoplasmicCa²⁺ concentration as a consequence of store depletion, and such a risein Ca²⁺ could open Ca²⁺-activated cation channels permeable to Ca²⁺. Oneway to avoid such problems is to use agents under conditions wherecytoplasmic Ca²⁺ has been strongly buffered with high concentrations ofCa²⁺ chelator such as EGTA or BAPTA.

Store-Operated Calcium Entry

Reduced calcium concentration in intracellular calcium stores such asthe endoplasmic reticulum resulting from release of calcium therefromprovides a signal for influx of calcium from the extracellular mediuminto the cell. This influx of calcium, which produces a sustained“plateau” elevation of cytosolic calcium concentration, generally doesnot rely on voltage-gated plasma membrane channels and does not involveactivation of calcium channels by calcium. This calcium influx mechanismis referred to as capacitive calcium entry (CCE), calciumrelease-activated, store-operated or depletion-operated calcium entry.Store-operated calcium entry can be recorded as an ionic current withdistinctive properties. This current is referred to as I_(SOC)(store-operated current) or I_(CAC) (calcium release-activated current).

Electrophysiological analysis of store-operated or calciumrelease-activated currents reveal distinct biophysical properties (see,e.g., Parekh and Penner (1997) Physiol. Rev. 77:901-930) of thesecurrents. For example, the current can be activated by depletion ofintracellular calcium stores (e.g., by non-physiological activators suchas thapsigargin, CPA, ionomycin and BAPTA, and physiological activatorssuch as IP₃) and can be selective for divalent cations, such as calcium,over monovalent ions in physiological solutions or conditions, can beinfluenced by changes in cytosolic calcium levels, and can show alteredselectivity and conductivity in the presence of low extracellularconcentrations of divalent cations. The current may also be blocked orenhanced by 2-APB (depending on concentration) and blocked by SKF96365and Gd³⁺ and generally can be described as a calcium current that is notstrictly voltage-gated.

Patch-clamp studies in mast cells and Jurkat leukemic T cells haveestablished the CRAC entry mechanism as an ion channel with distinctivebiophysical characteristics, including a high selectivity for Ca²⁺paired with an exceedingly low conductance. Furthermore, the CRACchannel was shown to fulfill the rigorous criteria for beingstore-operated, which is the activation solely by the reduction of Ca²⁺in the ER rather than by cytosolic Ca²⁺ or other messengers generated byPLC (Prakriya et al., In Molecular and Cellular Insights into IonChannel Biology (ed. Robert Maue) 121-140 (Elsevier Science, Amsterdam,2004)).

Regulation of Store-Operated Calcium Entry by Intracellular CalciumStores

Store-operated calcium entry is regulated by the level of calcium withinan intracellular calcium store. Intracellular calcium stores can becharacterized by sensitivity to agents, which can be physiological orpharmacological, which activate release of calcium from the stores orinhibit uptake of calcium into the stores. Different cells have beenstudied in characterization of intracellular calcium stores, and storeshave been characterized as sensitive to various agents, including, butnot limited to, IP₃ and compounds that effect the IP₃ receptor,thapsigargin, ionomycin and/or cyclic ADP-ribose (cADPR) (see, e.g.,Berridge (1993) Nature 361:315-325; Churchill and Louis (1999) Am. J.Physiol. 276:C₄26-C₄34: Dargie et al. (1990) Cell Regul. 1:279-290;Gerasimenko et al. (1996) Cell 84:473-480; Gromoda et al. (1995) FEBSLett. 360:303-306, Guse et al. (1999) Nature 398:70-73).

Accumulation of calcium within endoplasmic reticulum and sarcoplasmicreticulum (SR; a specialized version of the endoplasmic reticulum instriated muscle) storage organelles is achieved throughsarcoplasmic-endoplasmic reticulum calcium ATPases (SERCAs), commonlyreferred to as calcium pumps. During signaling (i.e., when endoplasmicreticulum channels are activated to provide for calcium release from theendoplasmic reticulum into the cytoplasm), endoplasmic reticulum calciumis replenished by the SERCA pump with cytoplasmic calcium that hasentered the cell from the extracellular medium (Yu and Hinkle (2000) J.Biol. Chem. 275:23648-23653; Hofer et al. (1998) EMBO J. 17:1986-1995).

Calcium release channels associated with IP₃ and ryanodine receptorsprovide for controlled release of calcium from endoplasmic andsarcoplasmic reticulum into the cytoplasm resulting in transientincreases in cytoplasmic calcium concentration. IP₃ receptor-mediatedcalcium release is triggered by IP₃ formed by the breakdown of plasmamembrane phosphoinositides through the action of phospholipase C, whichis activated by binding of an agonist to a plasma membrane Gprotein-coupled receptor or tyrosine kinase. Ryanodine receptor-mediatedcalcium release is triggered by an increase in cytoplasmic calcium andis referred to as calcium-induced calcium release (CICR). The activityof ryanodine receptors (which have affinity for ryanodine and caffeine)may also be regulated by cyclic ADP-ribose.

Thus, the calcium levels in the stores, and in the cytoplasm, fluctuate.For example, ER free calcium concentration can decrease from a range ofabout 60-400 μM to about 1-50 μM when HeLa cells are treated withhistamine, an agonist of PLC-linked histamine receptors (Miyawaki et al.(1997) Nature 388:882-887). Store-operated calcium entry is activated asthe free calcium concentration of the intracellular stores is reduced.Depletion of store calcium, as well as a concomitant increase incytosolic calcium concentration, can thus regulate store-operatedcalcium entry into cells.

Cytoplasmic Calcium Buffering

Agonist activation of signaling processes in cells can involve dramaticincreases in the calcium permeability of the endoplasmic reticulum, forexample, through opening of IP₃ receptor channels, and the plasmamembrane through store-operated calcium entry. These increases incalcium permeability are associated with an increase in cytosoliccalcium concentration that can be separated into two components: a“spike” of calcium release from the endoplasmic reticulum duringactivation of the IP₃ receptor and a plateau phase which is a sustainedelevation of calcium levels resulting from entry of calcium into thecytoplasm from the extracellular medium. Upon stimulation, the restingintracellular free calcium concentration of about 100 nM can riseglobally to greater than 1 μM and higher in microdomains of the cell.The cell modulates these calcium signals with endogenous calciumbuffers, including physiological buffering by organelles such asmitochondria, endoplasmic reticulum and Golgi. Mitochondrial uptake ofcalcium through a uniporter in the inner membrane is driven by the largenegative mitochondrial membrane potential, and the accumulated calciumis released slowly through sodium-dependent and -independent exchangers,and, under some circumstances, the permeability transition pore (PTP).Thus, mitochondria can act as calcium buffers by taking up calciumduring periods of cellular activation and can slowly release it later.Uptake of calcium into the endoplasmic reticulum is regulated by thesarcoplasmic and endoplasmic reticulum calcium ATPase (SERCA). Uptake ofcalcium into the Golgi is mediated by a P-type calcium transport ATPase(PMR₁/ATP2C₁). Additionally, there is evidence that a significant amountof the calcium released upon IP₃ receptor activation is extruded fromthe cell through the action of the plasma membrane calcium ATPase. Forexample, plasma membrane calcium ATPases provide the dominant mechanismfor calcium clearance in human T cells and Jurkat cells, althoughsodium/calcium exchange also contributes to calcium clearance in human Tcells.

Within calcium-storing organelles, calcium ions can be bound tospecialized calcium-buffering proteins, such as, for example,calsequestrins, calreticulins and calnexins. Additionally, there arecalcium-buffering proteins in the cytosol that modulate calcium spikesand assist in redistribution of calcium ions. Thus, proteins and othermolecules that participate in any of these and other mechanisms throughwhich cytosolic calcium levels can be reduced are proteins that areinvolved in, participate in and/or provide for cytoplasmic calciumbuffering. Thus, cytoplasmic calcium buffering helps regulatecytoplasmic Ca²⁺ levels during periods of sustained calcium influxthrough SOC channels or bursts of Ca²⁺ release. Large increases incytoplasmic Ca²⁺ levels or store refilling deactivate SOCE.

Downstream Calcium Entry-Mediated Events

In addition to intracellular changes in calcium stores, store-operatedcalcium entry affects a multitude of events that are consequent to or inaddition to the store-operated changes. For example Ca²⁺ influx resultsin the activation of a large number of calmodulin-dependent enzymesincluding the serine phosphatase calcineurin. Activation of calcineurinby an increase in intracellular calcium results in acute secretoryprocesses such as mast cell degranulation. Activated mast cells releasepreformed granules containing histamine, heparin, TNFα and enzymes suchas β-hexosaminidase. Some cellular events, such as B and T cellproliferation, require sustained calcineurin signaling, which requires asustained increase in intracellular calcium. A number of transcriptionfactors are regulated by calcineurin, including NFAT (nuclear factor ofactivated T cells), MEF₂ and NFκB. NFAT transcription factors playimportant roles in many cell types, including immune cells. In immunecells NFAT mediates transcription of a large number of molecules,including cytokines, chemokines and cell surface receptors.Transcriptional elements for NFAT have been found within the promotersof cytokines such as IL-2, IL-3, IL-4, IL-5, IL-8, IL-13, as well astumor necrosis factor alpha (TNFα), granulocyte colony-stimulatingfactor (G-CSF), and gamma-interferon (γ-IFN).

The activity of NFAT proteins is regulated by their phosphorylationlevel, which in turn is regulated by both calcineurin and NFAT kinases.Activation of calcineurin by an increase in intracellular calcium levelsresults in dephosphorylation of NFAT and entry into the nucleus.Rephosphorylation of NFAT masks the nuclear localization sequence ofNFAT and prevents its entry into the nucleus. Because of its strongdependence on calcineurin-mediated dephosphorylation for localizationand activity, NFAT is a sensitive indicator of intracellular freecalcium levels.

Calcium Channel Inhibitors

Disclosed herein are a number of Calcium channel inhibitors consistentwith the methods, compositions, administration regimens and compositionsfor use disclosed herein. In some embodiments a Calcium channelinhibitor is a SOC inhibitor. In some embodiments the Calcium channelinhibitor is a CRAC inhibitor. In some embodiments, the Calcium channelinhibitor inhibits a channel comprising STIM1 protein. In someembodiments, the Calcium channel inhibitor inhibits a channel comprisingOrai1 protein. In some embodiments, the Calcium channel inhibitorinhibits a channel comprising Orai2 protein.

In some embodiments the compound is a compound having the structure of:

or a pharmaceutically acceptable salt, pharmaceutically acceptablesolvate, or pharmaceutically acceptable prodrug thereof. In someembodiments the compound is selected form a list of compoundsconsisting:N-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamide.In some aspects the intracellular Calcium signaling inhibitor is acompound ofN-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamideor a pharmaceutically acceptable salt, pharmaceutically acceptablesolvate, or pharmaceutically acceptable prodrug thereof. In some aspectsthe intracellular Calcium signaling inhibitor is chosen from among thecompounds,N-(5-(6-ethoxy-4-methylpyridin-3-yl)pyrazin-2-yl)-2,6-difluorobenzamide,N-(5-(2-ethyl-6-methylbenzo[d]oxazol-5-yl)pyridin-2-yl)-3,5-difluoroisonicotinamide,N-(4-(1-ethyl-3-(thiazol-2-yl)-1H-pyrazol-5-yl)phenyl)-2-fluorobenzamide,N-(5-(1-ethyl-3-(triflouromethyl)-1H-pyrazol-5-yl)pyrazin-2-yl)-2,4,6-trifluorobenzamide,4-chloro-1-methyl-N-(4-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)phenyl)-1H-pyrazole-5-carboxamide,N-(4-(3-(difluoromethyl)-5-methyl-1H-pyrazol-1-yl)-3-fluorophenyl)-2,6-dIfluorobenzamide,N-(4-(3-(difluoromethyl)-5-methyl-1H-pyrazol-1-yl)-3-fluorophenyl)-2,4,6-trifluorobenzamide,N-(4-(3-(difluoromethyl)-1-methyl-1H-pyrazol-5-yl)-3-fluorophenyl)-2,4,6-trifluorobenzamide,4-chloro-N-(3-fluoro-4-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)phenyl)-1-methyl-1H-pyrazole-5-carboxamide,3-fluoro-4-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)-N-((3-methylisothiazol-4-yl)methyl)aniline,N-(5-(7-chloro-2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-6-yl)pyridin-2-yl)-2,6-difluorobenzamide,N-(2,6-difluorobenzyl)-5-(1-ethyl-3-(thiazol-2-yl)-1H-pyrazol-5-yl)pyrimidin-2-amine,3,5-difluoro-N-(3-fluoro-4-(3-methyl-1-(thiazol-2-yl)-1H-pyrazol-4-yl)phenyl)isonicotinamide,5-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)-N-(2,4,6-trifluorobenzyl)pyridin-2-amine,N-(5-(1-ethyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)pyridin-2-yl)-2,4,6-trifluorobenzamide,N-(5-(5-chloro-2-methylbenzo[d]oxazol-6-yl)pyrazin-2-yl)-2,6-difluorobenzamide,N-(5-(6-ethoxy-4-methylpyridin-3-yl)thiazol-2-yl)-2,3,6-trifluorobenzamide,N-(5-(1-ethyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)pyridin-2-yl)-2,3,6-trifluorobenzamide,2,3,6-trifluoro-N-(3-fluoro-4-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)phenyl)benzamide,2,6-difluoro-N-(4-(5-methyl-2-(trifluoromethyl)oxazol-4-yl)phenyl)benzamide,orN-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamideor a pharmaceutically acceptable salt, pharmaceutically acceptablesolvate, or pharmaceutically acceptable prodrug thereof.

Calcium Signaling and Pancreatic Health

Calcium signaling is central to healthy pancreatic activity. Foodstimulates the release of acetylcholine (ACh) and cholecyctokinin (CCK),which interact with Phospholipase C (PLC)-linked receptors on PancreaticAcinar Cells (PACs). In healthy PACs, ACh or CCK receptors triggerformation of IP₃, 1,4,5-inositol triphosphate, which diffuses to theapical region and stimulates IP₃ receptors on the Endoplasmic Reticulum(ER) to release Ca²⁺ in a controlled, pulsatile manner. Ca²⁺oscillations stimulate release of zymogens (pro-enzymes) into thepancreatic duct. Over time, the ER Ca²⁺ needs to be replenished, whichis accomplished by gentile activation of CRAC channels in thebasolateral region of the cell.

In certain situations (e.g., alcoholism or binge drinking, gall stones,etc.), fatty acid ethyl esters (FAEEs) formed from alcohol, or bileacids which accumulate due to gallstones diffuse into the PACs. Insidethe PACs, FAEEs and bile acids cause massive release of ER Ca²⁺ byactivating IP₃ receptors. Hyperstimulation of CCK receptors can alsoelicit robust Ca²⁺ release from ER stores. Emptying of Ca²⁺ stores leadsto hyperactivation of CRAC channels, causing excessive influx of Ca²⁺.The large Ca²⁺ influx causes release of enzymes from zymogen granules,and inappropriate activation of intracellular trypsin, which itself thenactivated other pancreatic digestive enzymes and initiates autodigestionand necrosis of the pancreas that can be blocked by a CRAC channelinhibitor such as Compound I, GSK-7975A,N-(5-(2,5-dimethylbenzo[d]oxazol-6-yl)thiazol-2-yl)-2,3,6-trifluorobenzamide(“Compound II”), or2,3,6-trifluoro-N-(3-fluoro-4-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)phenyl)benzamide(“Compound III”).

If unaddressed, the inappropriate release and activation of digestiveenzymes such as trypsin from zymogen granules can lead to autodigestionof pancreatic cells, leading to pancreatitis. As mentioned above, acuteor chronic pancreatitis can have a substantial negative effect on anindividual's health.

Symptoms and Causes of Pancreatitis

Pancreatitis, acute or chronic, is associated with severe upperabdominal or left upper quadrant burning pain radiating to the back,nausea, and vomiting that is worsened with eating. Depending on theseverity of the condition, internal bleeding may also occur. Bloodpressure, heart and respiratory rates are often elevated, althoughdehydration may lead to a decrease rather than an increase in bloodpressure. The abdomen is often tender but less so than the pain at thepancreas itself. Reflex bowel paralysis is commonly seen in pancreatitiscases, and fever or jaundice is not uncommon. Common symptoms and signsof pancreatitis include: severe epigastric pain (upper abdominal pain)radiating to the back, nausea, vomiting, loss of appetite, fever, chills(shivering), hemodynamic instability (which includes shock), tachycardia(rapid heartbeat), respiratory distress, and peritonitis.

Less commonly observed symptoms, indicative of severe disease, include anumber of medical ‘signs’ indicative of severe abdomina distress:Grey-Tumer's sign (hemorrhagic discoloration of the flanks), Cullen'ssign (hemorrhagic discoloration of the umbilicus), Pleural effusions(fluid in the bases of the pleural cavity), Grünwald sign (appearance ofecchymosis, large bruise, around the umbilicus due to local toxic lesionof the vessels), Karte's sign (pain or resistance in the zone where thehead of pancreas is located (in epigastrium, 6-7 cm above theumbilicus)), Kamenchik's sign (pain with pressure under the xiphoidprocess), Mayo-Robson's sign (pain while pressing at the top of theangle lateral to the Erector spinae muscles and below the left 12th rib(left costovertebral angle (CVA)) such as at Mayo-Robson's point (apoint on border of inner ⅔ with the external ⅓ of the line thatrepresents the bisection of the left upper abdominal quadrant, wheretenderness on pressure exists in disease of the pancreas. At this pointthe tail of pancreas is projected on the abdominal wall).

A person suffering from pancreatitis may demonstrate some, all, or fewto none of the above-mentioned symptoms. In some cases abdominal painmay be the sole symptom of the condition.

Chronic pancreatitis can lead to diabetes or pancreatic cancer. Defectsin the delivery of digestive enzymes such as trypsin may lead toimpaired digestion, leading to weight loss.

As many as eighty percent of cases of pancreatitis are caused by alcoholand gallstones. Gallstones are the single most common etiology of acutepancreatitis. Alcohol is the single most common etiology of chronicpancreatitis.

However, aside from alcohol and gall stones, there are a number ofadditional causes of pancreatitis. Some medications may be associatedwith pancreatitis. Examples of medications associated with pancreatitisinclude corticosteroids such as prednisolone, HIV drugs such asdidanosine and pentamidine, diuretics, anticonvulsants such as valproicacid, chemotherapeutic agents such as L-asparaginase and azathioprine,estrogen, medications that increase blood triglycerides, statins such ascholesterol-lowering statins, antihyperglycemic agents like metformin,and gliptins such as vildagliptin, sitagliptin, saxagliptin, andlinagliptin, tetracycline, sulfonamides, azathioprine, mercaptopurine,pentamidine, Grimethoprim-suilfamethoxazole, and salicylates. In somecases, drugs which are used to treat conditions associated withincreased events of pancreatitis may also be incidentally linked topancreatitis. Examples include statins in dyslipidemia and gliptins indiabetes. Additionally, some atypical antipsychotics such as clozapine,risperidone, and olanzapine may also be responsible for causingpancreatitis. This list is not exhaustive.

Non-pharmaceutical causes of pancreatitis are also known. For example,inherited forms of pancreatitis are known that result in the activationof trypsinogen within the pancreas, leading to autodigestion. Genesimplicated in heritable pancreatitis include Trypsin1, which codes fortrypsinogen, SPINK1, which codes for a trypsin inhibitor, and cysticfibrosis transmembrane conductance regulator.

Other common nonpharmaceutical causes of pancreatitis include trauma,mumps, autoimmune disease, high blood calcium, hypothermia, andundergoing endoscopic retrograde cholangiopancreatography (ERCP).Pancreas divisum is a common congenital malformation of the pancreasthat may underlie some recurrent cases. Penetrating ulcers are alsoassociated with pancreatitis. Diabetes mellitus type 2 is associatedwith a 2.8-fold higher risk of developing symptoms of pancreatitis.Additional conditions associated with pancreatitis include pancreaticcancer, pancreatic duct stones, vasculitis (inflammation of the smallblood vessels in the pancreas), coxsackie virus infection, andporphyria, particularly acute intermittent porphyria and erythropoieticprotoporphyria. Pregnancy is associated with pancreatitis in some cases.

Repeated marathon running, anorexia and bulemia, as well as fattynecrosis, cystic fibrosis, and scorpion venom are implicated in somepancreatitis cases.

A number of infectious agents are implicated in pancreatitis. Examplesinclude viral infection by viruses such as Cytomegalovirus, Hepatitis B,Herpes simplex virus, Mumps Rubulavirus, Varicella-zoster virus;Bacterial infection, such as by bacteria of the genera Legionella,Leptospira, Mycoplasma, or Salmonella; fungal infection such as by fungiof the genera Aspergillus; or parasitic infection by nematodes of thegenus Ascaris or by apicomplexan alvelolates of the generaCryptosporidium and Toxoplasma; among others.

The medical students' mnemonic ‘GETSMASHED’ is often used to remembersome of the common causes of Pancreatitis: G—Gall stones E—EthanolT—Trauma S—Steroids M—Mumps A—Autoimmune Pancreatitis S—Scorpion stingH—Hyperlipidaemia, Hypothermia, Hyperparathyroidism E—Endoscopicretrograde cholangiopancreatography D—Drugs commonly azathioprine,valproic acid.

Pancreatitis may also be idiopathic, in which case no cause isidentified.

Categorization of Pancreatitis

Pancreatitis, particularly acute pancreatitis, is often classified aseither ‘mild’. ‘moderate’, or ‘severe’ depending upon the predominantresponse to cell injury. These categories are all characterized bymisactivation of pancreatic zymogens such as trypsinogen inside thepancreas, often due to colocalization with the trypsinogen maturasecathepsin, which activates trypsinogen to trypsin. All three categoriesare characterized by inflammation and edema of the pancreas. Moderateand severe pancreatitis are further characterized by pancreatic necrosisand secondary injury to extra-pancreatic organs, with moderate acutepancreatitis patients suffering transient (<48 hour) organ failure,while severe acute pancreatitis patients have persistent (>48 hour)organ failure.

In response to the above-mentioned issues, the pancreas may directlysynthesize inflammatory mediators such as TNF-α and IL-1, associatedwith an inflammatory response and recruitment of neutrophils to thepancreas, or due to necrosis and leakage of cellular components,otherwise activate the immune system. The inflammatory response may leadto secondary manifestations of pancreatitis, such as hypovolemia fromcapillary permeability, acute respiratory distress syndrome,disseminated intravascular coagulations, renal failure, cardiovascularfailure, and gastrointestinal hemorrhage.

Acute pancreatitis (acute hemorrhagic pancreatic necrosis) may furtherbe characterized by acute inflammation and necrosis of pancreasparenchyma, focal enzymic necrosis of pancreatic fat, and vesselnecrosis (hemorrhage) resulting from intrapancreatic activation ofpancreatic enzymes. Lipase activation may produce necrosis of fat tissuein pancreatic interstitium and peripancreatic spaces as well as vesseldamage. Digestion of vascular walls results in thrombosis andhemorrhage. Inflammatory infiltrate is rich in neutrophils. Due to thepancreas lacking a capsule, the inflammation and necrosis can extend toinclude fascial layers in the immediate vicinity of the pancreas.

Chronic pancreatitis is a prolonged inflammation of the pancreas thatalters the organ's normal structure and function. It may be associatedwith episodes of acute pancreatitis or with persistent abdominal pain ordigestive defects. Chronic pancreatitis sufferers usually demonstratepersistent abdominal pain or malabsorption of the fats in foods. Painduring food uptake, particularly fatty or high-protein food uptake, isalso common. Weight loss, due to malabsorption of food uptake or to areduction in food uptake due to discomfort, is also common.

A common complication of chronic pancreatitis is diabetes.

Alcoholism, tobacco use, malnutrition, trauma, hypercalcemia, calcifiedstones, cystic fibrosis, and hereditary defects in trypsinogenprocessing and stability are commonly associated with chronicpancreatitis.

Chronic pancreatitis is typically diagnosed based on tests on pancreaticstructure and function. Serum amylase and lipase may or may not bemoderately elevated in cases of chronic pancreatitis, owing to theuncertain levels of productive cell damage. Elevated lipase is the morelikely found of the two. Amylase and lipase are nearly always foundelevated in the acute condition along with an elevated CRP inflammatorymarker that is broadly in line with the severity of the condition.

A secretin stimulation test is perhaps the most accurate functional testfor diagnosis of chronic pancreatitis. Impairment of bi-carbonateproduction early in chronic pancreatitis is used to identify persons inearly stages of disease (sensitivity of 95%). Additional tests used todetermine chronic pancreatitis are fecal elastase measurement in stool,serum trypsinogen, computed tomography (CT), ultrasound, EUS, MRI, ERCPand MRCP. Pancreatic calcification may be seen on abdominal X-rays, aswell as CT scans. Notably, however, ERCP and X-rays may trigger acutepancreatitis.

A number of additional tests are available to assay for chronicpancreatitis. Elevated serum bilirubin and alkaline phosphatase levelsmay indicate chronic pancreatitis, in some cases indicating stricturingof the common bile duct due to edema, fibrosis or cancer. Autoimmuneresponse-related chronic pancreatitis may be accompanied by elevationsin ESR, IgG4, rheumatoid factor, ANA and anti-smooth muscle antibody,assay of any of which may indicate chronic pancreatitis in a person. Aclassic symptom of chronic pancreatitis, steatorrhea or foodmalabsorption, may be diagnosed by two different studies: Sudan chemicalstaining of feces or fecal fat excretion of 7 grams or more over a 24 hrperiod on a 100 g fat diet. To check for pancreatic exocrinedysfunction, an exemplary sensitive and specific test is the measurementof fecal elastase, which may be done with a single stool sample, and avalue of less than 200 μg/g indicates pancreatic insufficiency.

A number of methods are known to evaluate the severity of pancreatitisin a person. Common tests include BISAP, Ranson's, APACHE-II, and CTSI.The BISAP test, for example, is based upon the following criteriaassessed in the first 24 hours after admission: blood urea nitrogen >25mg/dL (8.92 mmol/L): Impaired Mental Status, defined as: disorientation,lethargy, somnolence, coma or stupor; >2 Systemic Inflammatory ResponseSyndrome Criteria; Age >60: and Pleural Effusion Present. A positiveassessment on any of these criteria results in a ‘point’ in a totalscore ranging from 0 to 5. In some implementations of the test,mortality rates ranged from less than 1% in the lowest-risk group tomore than 20% in the highest-risk group.

A number of references discuss tests for pancreatitis severity, each ofwhich is hereby incorporated by reference: Wu B U, Johannes R S, Sun X,Tabak Y, Conwell D L, Banks P A. The early prediction of mortality inacute pancreatitis: a large population-based study. Gut, 2008 December;57(12):1698-703. doi: 10.1136/gut.2008.152702. Epub 2008 Jun. 2. PubMedPMID: 18519429: Papachristou G I, Muddana V, Yadav D, O'Connell M,Sanders M K, Slivka A, Whitcomb D C. Comparison of BISAP, Ranson's,APACHE-II, and CTSI scores in predicting organ failure, complications,and mortality in acute pancreatitis. Am J Gastroenterol. 2010 February;105(2):435-41: quiz 442. doi: 10.1038/ajg.2009.622. Epub 2009 Oct. 27.PubMed PMID: 19861954; and Gompertz M. FemAndez L, Lara I, Miranda J P,Mancilla C, Berger Z. [Bedside index for severity in acute pancreatitis(BISAP) score as predictor of clinical outcome in acute pancreatitis:retrospective review of 128 patients]. Rev Med Chil. 2012August:140(8):977-83. doi: 10.1590/50034-98872012000800002. Spanish.PubMed PMID: 23282769.

Therapeutic Amelioration of Pancreatitis

Disclosed herein are compositions and methods for the therapeuticamelioration of pancreatitis and symptoms thereof, such as through theadministration of a calcium channel inhibitor such as a CRAC inhibitor.In some embodiments the pancreatitis is acute pancreatitis. In someembodiments the pancreatitis is chronic pancreatitis. In someembodiments a method of ameliorating the symptoms of pancreatitis in aperson is disclosed. In some embodiments a method of ameliorating thesymptoms of pancreatitis in a person is disclosed comprising the stepsof identifying a person in need of amelioration of symptoms ofpancreatitis, and administering an intracellular Calcium signalinginhibitor to said person at a dose sufficient to ameliorate saidsymptoms.

The person may be identified using, for example, a common test forpancreatitis symptoms, such as BISAP, Ranson's, APACHE-II, and CTSI. Thetest may be BISAP. The test may be Ranson's. The test may be APACHE II.The test may be CTSI. In some embodiments a person is identified as aperson in need of amelioration of symptoms of pancreatitis by having aBISAP score of 5, 4, 3, 2, or 1. In some embodiments the person isidentified as having a BISAP score of 2. In some embodiments the personis identified as having a BISAP score of 3.

In some embodiments the person is identified as having a BISAP score of4. In some embodiments the person is identified as having a BISAP scoreof 5. In some embodiments a person is identified as having at least 1,2, 3, 4, 5, 6, 7, 8, 9, or more than 9 symptoms of pancreatitis, such asthe symptoms of pancreatitis disclosed herein. In some embodiments,rather than a person, the subject is a non-human mammal.

In some embodiments the symptoms are acute pancreatitis symptoms. Insome embodiments the symptoms are chronic pancreatitis symptoms.

The symptoms may comprise at least one of abdominal pain, increasedblood amylase levels, increased blood lipase levels, enlarged pancreas,nausea, vomiting, internal bleeding, bowel paralysis, fever, jaundice,weight loss, and elevated heart rate. The symptoms may comprisepremature digestive enzyme activation. The premature digestive enzymeactivation may, for example, occur in a pancreas of said person. In someembodiments the enzyme comprises trypsin.

In some embodiments, the intracellular Calcium signaling inhibitor is anSOC channel inhibitor. In some embodiments, the intracellular Calciumsignaling inhibitor is a CRAC channel inhibitor. In some embodiments,the CRAC channel inhibitor comprises Compound I. In some embodiments,the CRAC channel inhibitor comprises GSK-7975A. In some embodiments, theCRAC channel inhibitor comprises Compound II. In some embodiments, theCRAC channel inhibitor comprises Compound III. In some embodiments,ameliorating symptoms of pancreatitis further comprises administering apainkiller medication. In some embodiments, ameliorating symptoms ofpancreatitis further comprises administering a painkiller medicationwherein the painkiller medication comprises an opiate. In someembodiments, ameliorating symptoms of pancreatitis further comprisesadministering a painkiller medication wherein the painkiller medicationcomprises morphine. In some embodiments, ameliorating symptoms ofpancreatitis further comprises administering a painkiller medicationwherein the painkiller medication comprises fentanyl. In someembodiments, ameliorating symptoms of pancreatitis further comprisesadministering a painkiller medication wherein the painkiller medicationcomprises tramadol. In some embodiments, ameliorating symptoms ofpancreatitis further comprises administering a painkiller medicationwherein the painkiller medication comprises meperidine.

In some embodiments, the intracellular Calcium signaling inhibitor isdelivered to achieve a tissue level concentration that is equal to,about, or greater than the in vitro IC₅₀ value determined for thecompound. In some embodiments the Calcium signaling inhibitor isdelivered to achieve a tissue level concentration that is 1.5×, 2×, 3×,4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×,19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×,33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, 46×,47×, 48×, 49×, 50×, 51×, 52×, 53×, 54×, 55×, 56×, 57×, 58×, 59×, 60×,61×, 62×, 63×, 64×, 65×, 66×, 67×, 68×, 69×, 70×, 71×, 72×, 73×, 74×,75×, 76×, 77×, 78×, 79×, 80×, 81×, 82×, 83×, 84×, 85×, 86×, 87×, 88×,89×, 90×, 91×, 92×, 93×, 94×, 95×, 96×, 97×, 98×, 99×, 100×, or anynon-integer multiple ranging from 1× to 100× of the in vitro IC₅₀ valuedetermined for the compound.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that ranges from 1× to 100×, 2× to80×, 3× to 60×, 4× to 5×, 5× to 45×, 6× to 44×, 7× to 43×, 8× to 43×, 9×to 41×, or 10× to 40×, or any non-integer within said range, of the invitro IC₅₀ value determined for the compound.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that is 1 μM, 2 μM, 3 μM, 4 μM, 5μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46μM, 47 μM, 48 μM, 49 μM, 50 μM, 51 μM 52 μM, 53 μM, 54 μM, 55 μM, 56 μM,57 μM, 58 μM, 59 μM, 60 μM 61 μM, 62 μM, 63 μM, 64 μM, 65 μM, 66 μM, 67μM, 68 μM, 69 μM, 70 μM, 71 μM, 72 μM, 73 μM, 74 μM, 75 μM, 76 μM, 77μM, 78 μM, 79 μM, 80 μM, 81 μM, 82 μM, 83 μM, 84 μM, 85 μM, 86 μM, 87μM, 88 μM, 89 μM, 90 μM, 91 μM, 92 μM, 93 μM, 94 μM, 95 μM, 96 μM, 97μM, 98 μM, 99 μM, 100 μM, or any non-integer multiple ranging from about1 μM to about 100 μM.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that ranges from 1 μM to 100 μM, 2μM to 90 μM, 3 μM to 80 μM, 4 μM to 70 μM, 5 μM to 60 μM, 6 μM to 50 μM,7 μM to 40 μM, 8 μM to 30 μM, 9 μM to 20 μM, or 10 μM to 40 μM, or anyinteger or non-integer within said range.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that ranges from 9.5 μM to 10.5 μM9 μM to 11 μM, 8 μM to 12 μM, 7 μM to 13 μM, 5 μM to 15 μM, 2 μM to 20μM or 1 μM to 50 μM, or any integer or non-integer within said range.

In some embodiments amelioration of pancreatitis comprises reduction inseverity of at least one pancreatitis symptom. In some embodimentsamelioration of pancreatitis comprises reduction in severity of at leastone pancreatitis symptom such that said symptom no longer impacts thepreviously affected person. In some embodiments amelioration comprisesreduction of at least one symptom so that it has no effect on theperson. In some embodiments amelioration comprises a 10%, 20%, 30%, 40%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% reduction in said symptom. Insome embodiments, amelioration comprises reduction in severity of aplurality of symptoms, such as 2, 3, 4, 5, 6, 7, 8, 9, or more than 9symptoms, up to and including all symptoms, said reduction comprising a10%, 20%, 30%, 40%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% reductionin said symptoms.

In some embodiments amelioration comprises halting the progression ofpancreatitis such as acute pancreatitis or chronic pancreatitis. In someembodiments amelioration comprises halting the progression ofpancreatitis such as acute pancreatitis or chronic pancreatitis suchthat more severe symptoms such as organ failure, pancreas necrosis ordeath do not occur.

Prophylactic Amelioration of Acute and Chronic Pancreatitis

Disclosed herein are compositions and methods for the prophylacticamelioration of acute pancreatitis and symptoms thereof, such as throughthe administration of a calcium channel inhibitor such as a CRACinhibitor. In some embodiments a method of ameliorating the symptoms ofpancreatitis in a person is disclosed. In some embodiments a method ofameliorating the symptoms of pancreatitis in a person is disclosedcomprising the steps of identifying a person in need of prophylacticamelioration of symptoms of pancreatitis, and administering anintracellular Calcium signaling inhibitor to said person at a dosesufficient to prophylactically ameliorate said symptoms.

In some embodiments, the intracellular Calcium signaling inhibitor is aSOC channel inhibitor. In some embodiments, the intracellular Calciumsignaling inhibitor is a CRAC channel inhibitor. In some embodiments,the CRAC channel inhibitor comprises Compound I. In some embodiments,the CRAC channel inhibitor comprises GSK-7975A. In some embodiments, theCRAC channel inhibitor comprises Compound II. In some embodiments, theCRAC channel inhibitor comprises Compound III. In some embodiments,ameliorating symptoms of pancreatitis further comprises administering apainkiller medication such as an opiate. Morphine is an exemplarypainkiller in some embodiments.

In some embodiments, the intracellular Calcium signaling inhibitor isdelivered to achieve a tissue level concentration that is equal to,about, or greater than the in vitro IC₅₀ value determined for thecompound. In some embodiments the Calcium signaling inhibitor isdelivered to achieve a tissue level concentration that is 1.5×, 2×, 3×,4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×,19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×,33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, 46×,47×, 48×, 49×, 50×, 51×, 52×, 53×, 54×, 55×, 56×, 57×, 58×, 59×, 60×,61×, 62×, 63×, 64×, 65×, 66×, 67×, 68×, 69×, 70×, 71×, 72×, 73×, 74×,75×, 76×, 77×, 78×, 79×, 80×, 81×, 82×, 83×, 84×, 85×, 86×, 87×, 88×,89×, 90×, 91×, 92×, 93×, 94×, 95×, 96×, 97×, 98×, 99×, 100×, or anynon-integer multiple ranging from 1× to 100× of the in vitro IC₅₀ valuedetermined for the compound.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that ranges from 1× to 100×, 2× to80×, 3× to 60×, 4× to 50×, 5× to 45×, 6× to 44×, 7× to 43×, 8× to 43×,9× to 41×, or 10× to 40×, or any non-integer within said range, of thein vitro IC₅₀ value determined for the compound.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that is 1 μM 2 μM, 3 μM, 4 μM, 5μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36μM, 37 μM, 38 μM 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM 46 μM,47 μM, 48 μM, 49 μM, 50 μM, 51 μM, 52 μM, 53 μM, 54 μM, 55 μM, 56 μM, 57μM, 58 μM, 59 μM, 60 μM, 61 μM, 62 μM, 63 μM, 64 μM, 65 μM, 66 μM, 67μM, 68 μM, 69 μM, 70 μM, 71 μM, 72 μM, 73 μM, 74 μM, 75 μM, 76 μM, 77μM, 78 μM, 79 μM, 80 μM, 81 μM, 82 μM, 83 μM, 84 μM, 85 μM, 86 μM, 87μM, 88 μM, 89 μM, 90 μM, 91 μM, 92 μM, 93 μM, 94 μM, 95 μM, 96 μM, 97μM, 98 μM, 99 μM, 100 μM, or any non-integer multiple ranging from about1 μM to about 100 μM.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that ranges from 1 μM to 100 μM, 2μM to 90 μM, 3 μM to 80 μM, 4 μM to 70 μM, 5 μM to 60 μM, 6 μM to 50 μM,7 μM to 40 μM, 8 μM to 30 μM, 9 μM to 20 μM, or 10 μM to 40 μM, or anyinteger or non-integer within said range.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that ranges from 9.5 μM to 10.5 μM,9 μM to 11 μM, 8 μM to 12 μM, 7 μM to 13 μM, 5 μM to 15 μM, 2 μM to 20μM or 1 μM to 50 μM, or any integer or non-integer within said range.

In some embodiments the method comprises prophylactically amelioratingan acute pancreatitis symptom. In some embodiments the method comprisesprophylactically ameliorating a chronic pancreatitis symptom.

Prophylactically ameliorating a symptom of pancreatitis may comprisereducing the severity, likelihood of occurrence, or duration of at leastone symptom of pancreatitis. Prophylactically ameliorating a symptom ofpancreatitis may comprise reducing the severity, likelihood ofoccurrence, or duration of at least one symptom of pancreatitis up tothe point that said at least one symptom does not occur in the person.In some embodiments, prophylactically ameliorating a symptom ofpancreatitis may comprise reducing the severity, likelihood ofoccurrence, or duration of 2, 3, 4, 5, 6, 7, 8, 9, or more than 9symptoms of pancreatitis, up to and including reducing the severity,likelihood of occurrence, or duration of all symptoms of pancreatitis ina person, such as the symptoms of pancreatitis disclosed herein. In someembodiments, rather than a person, the subject is a non-human mammal.

In some embodiments the person is diagnosed as having a gall stone. Insome embodiments the person exhibits symptoms of having a gall stone,such as pain, for example intense pain, in the upper-right side of theabdomen, and/or nausea and vomiting, which may steadily increase forfrom approximately 30 minutes to several hours. A patient may alsoexperience referred pain between the shoulder blades or below the rightshoulder.

In some embodiments the person suffers from alcoholism. In someembodiments the person suffers from chronic alcohol use. In someembodiments the person has suffered from at least one instance of acutealcohol poisoning.

In some embodiments the person is subjected to a drug regimen comprisingadministration of at least one of a steroid such as a corticosteroid,prednisolone, an HIV drug, didanosine, pentamidine, a diuretic, valproicacid. L-asparaginase, azathioprine, estrogen, a statin such as acholesterol-lowering statin, an antihyperglycemic agent, metformin, aglipin such as vildagliptin and sitagliptin, an atypical antipsychotic,clozapine, risperidone, and olanzapine.

In some embodiments the person is identified as harboring an inheritedform of pancreatitis. In some embodiments the person harbors a mutantallele of Trypsin1 associated with inherited pancreatitis. In someembodiments the person harbors a trypsinogen enzyme variant associatedwith pancreatitis. In some embodiments the person harbors a mutantallele of SPINK1 associated with inherited pancreatitis. In someembodiments the person harbors a mutant allele of a cystic fibrosistransmembrane conductance regulator associated with inheritedpancreatitis.

In some embodiments the person has suffered at least one of high bloodcalcium, hypothermia, endoscopic retrograde cholangiopancreatography(ERCP), pancreas divisum, a congenital malformation of the pancreas,diabetes mellitus type 2, pancreatic cancer, pancreatic duct stones,vasculitis, inflammation of the small blood vessels in the pancreas,coxsackie virus infection, and porphyra, such as acute intermittentporphyria and erythropoietic protoporphyria.

In some embodiments a bodily health condition of said person has beenimpacted at least one of a gall stone, ethanol poisoning, alcoholism,trauma, mumps, an autoimmune disorder, a scorpion sting,hyperlipidaemia, hypothermia, hyperparathyroidism, and endoscopicretrograde cholangiopancreatography, azathioprine, and valproic acid.

In some embodiments a bodily health condition of said person has beenimpacted by at least one of a Coxsackie virus, a Cytomegalovirus, aHepatitis B virus, a Herpes simplex virus, Mumps, a Varicella-zostervirus, a Legionella bacterium, a Leptospira bacterium, a Mycoplasmabacterium, a Salmonella bacterium, an Aspergillus fungus, an Ascarisparasite, a Cryptosporidium cell and a Toxoplasma cell.

Combination Administration with a Drug or Drugs Associated withPancreatitis

Disclosed herein are compositions and administration regimens for thecombinatorial administration of a Calcium channel inhibitor and a drugassociated with pancreatitis. In some embodiments an administrationregimen comprises administration to an individual of a drug associatedwith a negative impact on pancreatic activity, and administration of anintracellular Calcium signaling inhibitor.

In some embodiments the drug associated with a negative impact onpancreatic activity is a drug is selected from the list consisting of: asteroid such as a corticosteroid, prednisolone, an HIV drug, didanosine,pentamidine, a diuretic, valproic acid, L-asparaginase, azathioprine,estrogen, a statin such as a cholesterol-lowering statin, anantihyperglycemic agent, metformin, a glipin such as vildagliptin andsitagliptin, an atypical antipsychotic, clozapine, risperidone, andolanzapine, azathioprine, and valproic acid.

In some embodiments the intracellular Calcium signaling inhibitor is anSOC inhibitor. In some embodiments the intracellular Calcium signalinginhibitor is a CRAC inhibitor. An exemplary CRAC inhibitor comprisesCompound I. An exemplary CRAC inhibitor comprises GSK-7975A. Anexemplary CRAC inhibitor comprises Compound II. An exemplary CRACinhibitor comprises Compound III.

In some embodiments the administration regimen comprises administrationof a calcium channel inhibitor such as a CRAC inhibitor such as at leastone of Compound I, GSK 7975A, Compound II, and Compound III in concertwith a drug associated with a negative impact on pancreatic activity. Insome embodiments the calcium channel inhibitor such as a CRAC inhibitorsuch as at least one of Compound I, GSK 7975A, Compound II, and CompoundIII is administered on the same day as a drug associated with a negativeimpact on pancreatic activity.

In some embodiments the calcium channel inhibitor such as a CRACinhibitor such as at least one of Compound I, GSK 7975A, Compound II,and Compound III is administered on the same week as a drug associatedwith a negative impact on pancreatic activity. In some embodiments thecalcium channel inhibitor such as a CRAC inhibitor such as at least oneof Compound I, GSK 7975A, Compound II, and Compound III is administeredconcurrently with each administration of a drug associated with anegative impact on pancreatic activity. In some embodiments the calciumchannel inhibitor such as a CRAC inhibitor such as at least one ofCompound I, GSK 7975A, Compound II, and Compound III is administered onan administration regimen pattern that is independent of theadministration pattern for a drug associated with a negative impact onpancreatic activity. In some embodiments the calcium channel inhibitorsuch as a CRAC inhibitor such as at least one of Compound I, GSK 7975A,Compound II, and Compound III is administered through the same route ofdelivery, such as orally or intravenously, as a drug associated with anegative impact on pancreatic activity. In some embodiments the calciumchannel inhibitor such as a CRAC inhibitor such as at least one ofCompound I, GSK 7975A, Compound II, and Compound III is administeredthrough a separate route of delivery compared to a drug associated witha negative impact on pancreatic activity. In some embodiments thecalcium channel inhibitor such as a CRAC inhibitor such as at least oneof Compound I, GSK 7975A, Compound II, and Compound III is administeredto a person receiving a drug associated with a negative impact onpancreatic activity only after said person shows at least one sign of animpact of said drug on pancreatic activity, for example through anincrease in blood amylase activity or blood lipase activity, or throughmanifestation of at least one pancreatitis symptom as disclosed herein.In some embodiments the calcium channel inhibitor such as a CRACinhibitor such as at least one of Compound I, GSK 7975A, Compound II,and Compound III is administered to a person receiving a drug associatedwith a negative impact on pancreatic activity in the absence of anyevidence in or from said person related to any sign of an impact of saiddrug on pancreatic activity, for example through an increase in bloodamylase activity or blood lipase activity, or through manifestation ofat least one pancreatitis symptom as disclosed herein.

In some embodiments the calcium channel inhibitor such as a CRACinhibitor such as at least one of Compound I, GSK 7975A, Compound II,and Compound III is administered in a single composition with a drugassociated with a negative impact on pancreatic activity. Accordingly,some embodiments disclosed herein relate to a composition comprising anintracellular Calcium signaling inhibitor and at least one drugassociated with a negative impact on pancreatic activity. In someembodiments the at least one drug selected from the list consisting of:a steroid such as a corticosteroid, prednisolone, an HIV drug,didanosine, pentamidine, a diuretic, valproic acid, L-asparaginase,azathioprine, estrogen, a statin such as a cholesterol-lowering statin,an antihyperglycemic agent, metformin, a glipin such as vildagliptin andsitagliptin, an atypical antipsychotic, clozapine, risperidone, andolanzapine, azathioprine, and valproic acid.

In some embodiments, the intracellular Calcium signaling inhibitor ofsaid composition is an SOC inhibitor. In some embodiments, theintracellular Calcium signaling inhibitor is a CRAC inhibitor. In someembodiments, said CRAC inhibitor comprises Compound I. In someembodiments, said CRAC inhibitor comprises GSK-7975A. In someembodiments, said CRAC inhibitor comprises Compound II. In someembodiments, said CRAC inhibitor comprises Compound III.

In some embodiments, the intracellular Calcium signaling inhibitor isdelivered to achieve a tissue level concentration that is equal to,about, or greater than the in vitro IC₅₀ value determined for thecompound. In some embodiments the Calcium signaling inhibitor isdelivered to achieve a tissue level concentration that is 1.5×, 2×, 3×,4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×,19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×,33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, 46×,47×, 48×, 49×, 50×, 51×, 52×, 53×, 54×, 55×, 56×, 57×, 58×, 59×, 60×,61×, 62×, 63×, 64×, 65×, 66×, 67×, 68×, 69×, 70×, 71×, 72×, 73×, 74×,75×, 76×, 77×, 78×, 79×, 80×, 81×, 82×, 83×, 84×, 85×, 86×, 87×, 88×,89×, 90×, 91×, 92×, 93×, 94×, 95×, 96×, 97×, 98×, 99×, 100×, or anynon-integer multiple ranging from 1× to 10× of the in vitro IC₅₀ valuedetermined for the compound.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that ranges from 1× to 100×, 2× to80×, 3× to 60×, 4× to 50×, 5× to 45×, 6× to 44×, 7× to 43×, 8× to 43×,9× to 41×, or 10× to 40×, or any non-integer within said range, of thein vitro IC₅₀ value determined for the compound.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that is 1 μM, 2 μM, 3 μM, 4 μM, 5μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM 26 μM,27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM 33 μM, 34 μM, 35 μM, 36 μM, 37μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47μM, 48 μM, 49 μM, 50 μM, 51 μM, 52 μM, 53 μM, 54 μM, 55 μM, 56 μM, 57μM, 58 μM, 59 μM, 60 μM, 61 μM, 62 μM, 63 μM, 64 μM, 65 μM, 66 μM, 67μM, 68 μM, 69 μM, 70 μM, 71 μM, 72 μM, 73 μM, 74 μM, 75 μM, 76 μM, 77μM, 78 μM, 79 μM, 80 μM, 81 μM, 82 μM, 83 μM, 84 μM, 85 μM, 86 μM, 87μM, 88 μM, 89 μM, 90 μM, 91 μM, 92 μM, 93 μM, 94 μM, 95 μM, 96 μM, 97μM, 98 μM, 99 μM, 100 μM, or any non-integer multiple ranging from about1 μM to about 100 μM.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that ranges from 1 μM to 100 μM, 2μM to 90 μM, 3 μM to 80 μM, 4 μM to 70 μM, 5 μM to 60 μM, 6 μM to 50 μM,7 μM to 40 μM, 8 μM to 30 μM, 9 μM to 20 μM, or 10 μM to 40 μM, or anyinteger or non-integer within said range.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that ranges from 9.5 μM to 10.5 μM,9 μM to 11 μM, 8 μM to 12 μM, 7 μM to 13 μM, 5 μM to 15 μM, 2 μM to 20μM or 1 μM to 50 μM, or any integer or non-integer within said range.

In some embodiments, the composition additionally comprises at least oneof an excipient, a solubilizer, a surfactant, a disintegrant, and abuffer. In some embodiments the composition is a liquid or an emulsion.In some embodiments the composition is a liquid, a nanoparticle, ananoparticle suspension, or a nanoparticle emulsion. In some embodimentsthe composition is a tablet.

Calcium Signaling and Viral Diseases

A viral disease occurs when an organism's body (the host) is invaded bypathogenic viruses. Infectious virus particles, called virions, attachto and enter susceptible cells of the host. Calcium signaling modulatesviral entry, production, and transmission in a host cell, therebyspreading the viral disease. By way of example, a host cell's calciumsignaling is triggered by the viral disease through activation of STIM1-and Orai-mediated calcium entry, which further allows the virus to budand replicate within the host cell.

Viral diseases are diverse and categorized by structuralcharacteristics, such as genome type, virion shape, replication site,etc. By way of a specific non-limiting example, a viral diseasecomprises a hemorrhagic fever virus. In some aspects, a hemorrhagicfever virus is an arenavirus, a filovirus, a bunyavirus, a flavivirus, arhabdovirus, or combinations thereof.

Hemorrhagic fever viruses include, by way of non-limiting examples,Ebola virus, Marburg virus, Lassa virus, Junin virus, Rotavirus, WestNile virus, Zika virus, Coxsackievirus, Hepatitis B virus, Epstein Barrvirus, dengue virus, Rift Valley virus, etc.

Disclosed herein are compositions and methods for the prophylacticamelioration of a viral disease and symptoms thereof, such as throughthe administration of a calcium channel inhibitor such as a CRACinhibitor. In some embodiments a method of ameliorating the symptoms ofa viral disease in a person is disclosed. In some embodiments a methodof ameliorating the symptoms of a viral disease in a person is disclosedcomprising the steps of identifying a person in need of prophylacticamelioration of symptoms of a viral disease, and administering anintracellular Calcium signaling inhibitor to said person at a dosesufficient to prophylactically ameliorate said symptoms.

In some embodiments, the common symptoms of a viral disease comprisefever or bleeding diathesis. In further embodiments, the symptoms of aviral disease comprise flushing of the face, flushing of the chest,petechiae, capillary leak, bleeding, swelling, edema, hypotension,shock, or combinations thereof. In even further embodiments, thesymptoms of a viral disease comprise malaise, muscle pain, headache,vomiting, diarrhea, or combinations thereof.

In some embodiments, the intracellular Calcium signaling inhibitor is aSOC channel inhibitor. In some embodiments, the intracellular Calciumsignaling inhibitor is a CRAC channel inhibitor. In some embodiments,the CRAC channel inhibitor comprises Compound I. In some embodiments,the CRAC channel inhibitor comprises GSK-7975A. In some embodiments, theCRAC channel inhibitor comprises Compound II. In some embodiments, theCRAC channel inhibitor comprises Compound III. In some embodiments,ameliorating symptoms of a viral disease further comprises administeringan antiviral medication or a vaccine.

In some embodiments, the intracellular Calcium signaling inhibitor isdelivered to achieve a tissue level concentration that is equal to,about, or greater than the in vitro ICs value determined for thecompound. In some embodiments the Calcium signaling inhibitor isdelivered to achieve a tissue level concentration that is 1.5×, 2×, 3×,4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×,19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×,33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, 46×,47×, 48×, 49×, 50×, 51×, 52×, 53×, 54×, 55×, 56×, 57×, 58×, 59×, 60×,61×, 62×, 63×, 64×, 65×, 66×, 67×, 68×, 69×, 70×, 71×, 72×, 73×, 74×,75×, 76×, 77×, 78×, 79×, 80×, 81×, 82×, 83×, 84×, 85×, 86×, 87×, 88×,89×, 90×, 91×, 92×, 93×, 94×, 95×, 96×, 97×, 98×, 99×, 100×, or anynon-integer multiple ranging from 1× to 100× of the in vitro ICS valuedetermined for the compound.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that ranges from 1× to 100×, 2× to80×, 3× to 60×, 4× to 50×, 5× to 45×, 6× to 44×, 7× to 43×, 8× to 43×,9× to 41×, or 10× to 40×, or any non-integer within said range, of thein vitro IC₅₀ value determined for the compound.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that is 1 μM, 2 μM, 3 μM, 4 μM, 5μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11μM, 12 μM, 13 μM, 14 μM, 15 μM, 16μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46μM, 47 μM, 48 μM 49 μM, 50 μM, 51 μM, 52 μM, 53 μM, 54 μM, 55 μM, 56 μM,57 μM, 58 μM, 59 μM, 60 μM, 61 μM, 62 μM, 63 μM, 64 μM, 65 μM, 66 μM, 67μM, 68 μM, 69 μM, 70 μM, 71 μM, 72 μM, 73 μM, 74 μM, 75 μM, 76 μM, 77μM, 78 μM, 79 μM, 80 μM, 81 μM, 82 μM, 83 μM, 84 μM, 85 μM, 86 μM, 87μM, 88 μM, 89 μM, 90 μM, 91 μM, 92 μM, 93 μM, 94 μM, 95 μM, 96 μM, 97μM, 98 μM, 99 μM, 100 μM, or any non-integer multiple ranging from about1 μM to about 100 μM.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that ranges from 1 μM to 100 μM, 2μM to 90 μM, 3 μM to 80 μM, 4 μM to 70 μM, 5M to 60 μM, 6 μM to 50 μM, 7μM to 40 μM, 8 μM to 30 μM, 9 μM to 20 μM, or 10 μM to 40 μM, or anyinteger or non-integer within said range.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that ranges from 9.5 μM to 10.5 μM,9 μM to 11 μM, 8 μM to 12 μM, 7 μM to 13 μM 5 μM to 15 μM, 2 μM to 20 μMor 1 μM to 50 μM, or any integer or non-integer within said range.

In some embodiments the method comprises prophylactically amelioratingan acute viral disease symptom. In some embodiments the method comprisesprophylactically ameliorating a chronic viral disease symptom.

Prophylactically ameliorating a symptom of a viral disease comprisereducing the severity, likelihood of occurrence, or duration of at leastone symptom of the viral disease. Prophylactically ameliorating asymptom of a viral disease comprise reducing the severity, likelihood ofoccurrence, or duration of at least one symptom of the viral disease upto the point that said at least one symptom does not occur in theperson. In some embodiments, prophylactically ameliorating a symptom ofa viral disease comprise reducing the severity, likelihood ofoccurrence, or duration of 2, 3, 4, 5, 6, 7, 8, 9, or more than 9symptoms of a viral disease, up to and including reducing the severity,likelihood of occurrence, or duration of all symptoms of the viraldisease in a person, such as the symptoms of viral diseases disclosedherein. In some embodiments, rather than a person, the subject is anon-human mammal.

Calcium Signaling and Th17-Induced Diseases

T helper cells (Th cells) are critical to immune system functioning. Thcells regulate the immune system by releasing T cell cytokines,comprising chemokines, interferons, interleukins, lymphokines, tumornecrosis factor, or combinations thereof. T helper 17 cells (Th17) areasubset of pro-inflammatory Th cells and are defined by their productionof interleukin 17 (IL-17). Dysregulation of Th17 is associated withinflammatory and autoimmune disorders. Calcium signaling plays a crucialrole in modulating Th17 differentiation.

Disclosed herein are compositions and methods for the prophylacticamelioration of Th17-induced disease and symptoms thereof, such asthrough the administration of a calcium channel inhibitor such as a CRACinhibitor. In some embodiments a method of ameliorating the symptoms ofTh17-induced diseases in a person is disclosed. In some embodiments amethod of ameliorating the symptoms of Th17-induced disease in a personis disclosed comprising the steps of identifying a person in need ofprophylactic amelioration of symptoms of a Th17-induced disease, andadministering an intracellular Calcium signaling inhibitor to saidperson at a dose sufficient to prophylactically ameliorate saidsymptoms.

In some embodiments, the symptoms of Th17-induced disease comprise acuteinflammation. Symptoms of inflammation in a person comprise localizedreddening, swelling, heat, pain, stiffness, fever, chills, fatigue,headache, appetite loss, or combinations thereof. In some aspects, thesymptoms occur on the body of the person, including the torso, arms,hands, fingers, legs, feet, toes, head, neck, bones, joints, throat,sinuses, eyes, or combinations thereof.

In other embodiments, Th17-induced disease comprises chronicinflammation or a chronic inflammatory disease. Chronic inflammatorydiseases, by way of non-limiting examples, include hay fever,periodontitis, atherosclerosis, rheumatoid arthritis, or cancer.

In even further embodiments, Th17-induced diseases comprise anautoimmune disease. Autoimmune diseases are diseases in which the body'simmune system attacks healthy cells. Autoimmune diseases occur in theheart, kidney, liver, lung, skin, endocrine glands, exocrine glands,digestive system, tissue, blood, nervous system, or vascular system. Byway of non-limiting examples, autoimmune diseases include rheumatoidarthritis, lupus, celiac disease, psoriasis, Sjorgen's syndrome,polymyalgia rheumatica, multiple sclerosis, ankylosing spondylitis, type1 diabetes, alopecia areata, vasculitis, temporal arteritis, etc.

In some embodiments, the intracellular Calcium signaling inhibitor is aSOC channel inhibitor. In some embodiments, the intracellular Calciumsignaling inhibitor is a CRAC channel inhibitor. In some embodiments,the CRAC channel inhibitor comprises Compound I. In some embodiments,the CRAC channel inhibitor comprises GSK-7975A. In some embodiments, theCRAC channel inhibitor comprises Compound II. In some embodiments, theCRAC channel inhibitor comprises Compound III. In some embodiments,ameliorating symptoms of Th17-induced diseases further comprisesadministering an anti-inflammatory medication.

In some embodiments, the intracellular Calcium signaling inhibitor isdelivered to achieve a tissue level concentration that is equal to,about, or greater than the in vitro IC₅₀ value determined for thecompound. In some embodiments the Calcium signaling inhibitor isdelivered to achieve a tissue level concentration that is 1.5×, 2×, 3×,4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×,19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×,33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, 46×,47×, 48×, 49×, 50×, 51×, 52×, 53×, 54×, 55×, 56×, 57×, 58×, 59×, 60×,61×, 62×, 63×, 64×, 65×, 66×, 67×, 68×, 69×, 70×, 71×, 72×, 73×, 74×,75×, 76×, 77×, 78×, 79×, 80×, 81×, 82×, 83×, 84×, 85×, 86×, 87×, 88×,89×, 90×, 91×, 92×, 93×, 94×, 95×, 96×, 97×, 98×, 99×, 100×, or anynon-integer multiple ranging from 1× to 100× of the in vitro IC₅₀ valuedetermined for the compound.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that ranges from 1× to 100×, 2× to80×, 3× to 60×, 4× to 50×, 5× to 45×, 6× to 44×, 7× to 43×, 8× to 43×,9× to 41×, or 10× to 40×, or any non-integer within said range, of thein vitro IC₅₀ value determined for the compound.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that is 1 μM, 2 μM, 3 μM, 4 μM, 5μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46μM, 47 μM, 48 μM, 49 μM, 50 μM, 51 μM, 52 μM, 53 μM, 54 μM, 55 μM, 56μM, 57 μM, 58 μM, 59 μM, 60 μM, 61 μM, 62 μM, 63 μM, 64 μM, 65 μM, 66μM, 67 μM, 68 μM, 69 μM, 70 μM, 71 μM, 72 μM, 73 μM 74 μM, 75 μM, 76 μM,77 μM, 78 μM, 79 μM, 8 μM, 8 μM, 82 μM, 83 μM, 84 μM, 85 μM, 86 μM, 87μM, 88 μM, 89 μM, 90 μM, 91 μM, 92 μM, 93 μM, 94 μM, 95 μM, 96 μM, 97μM, 98 μM, 99 μM, 100 μM, or any non-integer multiple ranging from about1 μM to about 100 μM.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that ranges from 1 μM to 100 μM, 2μM to 90 μM, 3 μM to 80 μM, 4 μM to 70 μM, 5 μM to 60 μM, 6 μM to 50 μM,7 μM to 40 μM, 8 μM to 30 μM, 9 μM to 2 μM, or 10 μM to 40 μM or anyinteger or non-integer within said range.

In some embodiments the Calcium signaling inhibitor is delivered toachieve a tissue level concentration that ranges from 9.5 μM to 10.5 μM,9 μM to 11 μM, 8 μM to 12 μM, 7 μM to 13 μM, 5 μM to 15 μM, 2 μM to 20μM or 1 μM to 50 μM, or any integer or non-integer within said range.

In some embodiments the method comprises prophylactically amelioratingan acute Th17-induced disease symptom. In some embodiments the methodcomprises prophylactically ameliorating a chronic Th17-induced diseasesymptom.

Prophylactically ameliorating a symptom of Th17-induced diseasescomprise reducing the severity, likelihood of occurrence, or duration ofat least one symptom of the Th17-induced disease. Prophylacticallyameliorating a symptom of Th17-induced diseases comprise reducing theseverity, likelihood of occurrence, or duration of at least one symptomof the Th7-induced disease up to the point that said at least onesymptom does not occur in the person. In some embodiments,prophylactically ameliorating a symptom of Th17-induced diseasescomprise reducing the severity, likelihood of occurrence, or duration of2, 3, 4, 5, 6, 7, 8, 9, or more than 9 symptoms of a Th17-induceddisease, up to and including reducing the severity, likelihood ofoccurrence, or duration of all symptoms of the Th17-induced disease in aperson, such as the symptoms of Th17-induced diseases disclosed herein.In some embodiments, rather than a person, the subject is a non-humanmammal.

Certain Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood to which the claimedsubject matter pertains. In the event that there are a plurality ofdefinitions for terms herein, those in this section prevail. Allpatents, patent applications, publications and published nucleotide andamino acid sequences (e.g., sequences available in GenBank or otherdatabases) referred to herein are incorporated by reference. Wherereference is made to a URL or other such identifier or address, it isunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information can be foundby searching the internet. Reference thereto evidences the availabilityand public dissemination of such information.

It is to be understood that the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive of any subject matter claimed. In this application,the use of the singular includes the plural unless specifically statedotherwise. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. In thisapplication, the use of “or” means “and/or” unless stated otherwise.Furthermore, use of the term “including” as well as other forms, such as“include”, “includes,” and “included,” is not limiting.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Definition of standard chemistry terms may be found in reference works,including but not limited to, Carey and Sundberg “Advanced OrganicChemistry 4th Ed.” Vols. A (2000) and B (2001), Plenum Press, New York.Unless otherwise indicated, conventional methods of mass spectroscopy,NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniquesand pharmacology.

Unless specific definitions are provided, the nomenclature employed inconnection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those recognized in thefield. Standard techniques can be used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients. Standard techniques can be used for recombinantDNA, oligonucleotide synthesis, and tissue culture and transformation(e.g., electroporation, lipofection). Reactions and purificationtechniques can be performed e.g., using kits of manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures can be generallyperformed of conventional methods and as described in various generaland more specific references that are cited and discussed throughout thepresent specification.

It is to be understood that the methods and compositions describedherein are not limited to the particular methodology, protocols, celllines, constructs, and reagents described herein and as such may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the methods, compounds, compositions describedherein.

The terms “kit” and “article of manufacture” are used as synonyms.

The term “subject” or “patient” encompasses mammals and non-mammals.Examples of mammals include, but are not limited to, any member of theMammalian class: humans, non-human primates such as chimpanzees, andother apes and monkey species: farm animals such as cattle, horses,sheep, goats, swine; domestic animals such as rabbits, dogs, and cats;laboratory animals including rodents, such as rats, mice and guineapigs, and the like. Examples of non-mammals include, but are not limitedto, birds, fish and the like. In one embodiment of the methods andcompositions provided herein, the mammal is a human.

The terms “treat,” “treating” or “treatment,” as used herein, includealleviating, abating or ameliorating a disease or condition symptoms,preventing additional symptoms, ameliorating or preventing theunderlying causes of symptoms, inhibiting the disease or condition,e.g., arresting the development of the disease or condition, relievingthe disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition eitherprophylactically and/or therapeutically.

As used herein, the term “target protein” refers to a protein or aportion of a protein capable of being bound by, or interacting with acompound described herein, such as a compound with a structure from thegroup of Compound A. In certain embodiments, a target protein is a STIMprotein. In certain embodiments, a target protein is an Orai protein.

As used herein, “STIM protein” includes but is not limited to, mammalianSTIM-1, such as human and rodent (e.g., mouse) STIM-1, Drosophilamelanogaster D-STIM, C. elegans C-STIM, Anopheles gambiae STIM andmammalian STIM-2, such as human and rodent (e.g., mouse) STIM-2. (seeparagraphs 102111 through 10270 of US 2007/0031814, as well as Table 3of US 2007/0031814, herein incorporated by reference) As describedherein, such proteins have been identified as being involved in,participating in and/or providing for store-operated calcium entry ormodulation thereof, cytoplasmic calcium buffering and/or modulation ofcalcium levels in or movement of calcium into, within or out ofintracellular calcium stores (e.g., endoplasmic reticulum).

As used herein, an “Orai protein” includes Orai1 (SEQ ID NO: 1 asdescribed in WO 07/081804), Orai2 (SEQ ID NO: 2 as described in WO07/081804), or Orai3 (SEQ ID NO: 3 as described in WO 07/081804). Orai1nucleic acid sequence corresponds to GenBank accession number NM_032790,Orai2 nucleic acid sequence corresponds to GenBank accession numberBC069270 and Orai3 nucleic acid sequence corresponds to GenBankaccession number NM_152288. As used herein, Orai refers to any one ofthe Orai genes, e.g., Orai1, Orai2, Orai3 (see Table I of WO 07/081804).As described herein, such proteins have been identified as beinginvolved in, participating in and/or providing for store-operatedcalcium entry or modulation thereof, cytoplasmic calcium bufferingand/or modulation of calcium levels in or movement of calcium into,within or out of intracellular calcium stores (e.g., endoplasmicreticulum).

The term “fragment” or “derivative” when referring to a protein (e.g.STIM, Orai) means proteins or polypeptides which retain essentially thesame biological function or activity in at least one assay as the nativeprotein(s). For example, the fragments or derivatives of the referencedprotein maintains at least about 50% of the activity of the nativeproteins, at least 75%, at least about 95% of the activity of the nativeproteins, as determined e.g. by a calcium influx assay.

As used herein, amelioration of the symptoms of a particular disease,disorder or condition by administration of a particular compound orpharmaceutical composition refers to any lessening of severity, delay inonset, slowing of progression, or shortening of duration, whetherpermanent or temporary, lasting or transient that can be attributed toor associated with administration of the compound or composition.

The term “modulate,” as used herein, means to interact with a targetprotein either directly or indirectly so as to alter the activity of thetarget protein, including, by way of example only, to inhibit theactivity of the target, or to limit or reduce the activity of thetarget.

As used herein, the term “modulator” refers to a compound that alters anactivity of a target. For example, a modulator can cause an increase ordecrease in the magnitude of a certain activity of a target compared tothe magnitude of the activity in the absence of the modulator. Incertain embodiments, a modulator is an inhibitor, which decreases themagnitude of one or more activities of a target. In certain embodiments,an inhibitor completely prevents one or more activities of a target.

As used herein. “modulation” with reference to intracellular calciumrefers to any alteration or adjustment in intracellular calciumincluding but not limited to alteration of calcium concentration in thecytoplasm and/or intracellular calcium storage organelles, e.g.,endoplasmic reticulum, and alteration of the kinetics of calcium fluxesinto, out of and within cells. In aspect, modulation refers toreduction.

As used herein, the term “target activity” refers to a biologicalactivity capable of being modulated by a modulator. Certain exemplarytarget activities include, but are not limited to, binding affinity,signal transduction, enzymatic activity, tumor growth, inflammation orinflammation-related processes, and amelioration of one or more symptomsassociated with a disease or condition.

The terms “inhibits”. “inhibiting”, or “inhibitor” of SOC channelactivity or CRAC channel activity, as used herein, refer to inhibitionof store operated calcium channel activity or calcium release activatedcalcium channel activity.

The term “acceptable” with respect to a formulation, composition oringredient, as used herein, means having no persistent detrimentaleffect on the general health of the subject being treated.

The term “pharmaceutically acceptable,” as used herein, refers amaterial, such as a carrier, diluent, or formulation, which does notabrogate the biological activity or properties of the compound, and isrelatively nontoxic, i.e., the material may be administered to anindividual without causing undesirable biological effects or interactingin a deleterious manner with any of the components of the composition inwhich it is contained.

The term “pharmaceutical combination” as used herein, means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that one activeingredient, e.g. a compound with a structure from the group of CompoundA

and a co-agent, are administered to a patient as separate entitieseither simultaneously, concurrently or sequentially with no specificintervening time limits, wherein such administration provides effectivelevels of the two compounds in the body of the patient. The latter alsoapplies to cocktail therapy, e.g. the administration of three or moreactive ingredients.

The term “pharmaceutical composition” refers to a mixture of a compoundwith a structure from the group of Compound A, described herein withother chemical components, such as carriers, stabilizers, diluents,surfactants, dispersing agents, suspending agents, thickening agents,and/or excipients. The pharmaceutical composition facilitatesadministration of the compound to an organism. Multiple techniques ofadministering a compound exist in the art including, but not limited to:intravenous, oral, aerosol, parenteral, ophthalmic, subcutaneous,intramuscular, pulmonary and topical administration.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of an agent or a compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disease or condition being treated. The result can bereduction and/or alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. Forexample, an “effective amount” for therapeutic uses is the amount of thecomposition that includes a compound with a structure from the group ofCompound A, required to provide a clinically significant decrease indisease symptoms. An appropriate “effective” amount in any individualcase may be determined using techniques, such as a dose escalationstudy.

The terms “enhance” or “enhancing,” as used herein, means to increase orprolong either in potency or duration a desired effect. Thus, in regardto enhancing the effect of therapeutic agents, the term “enhancing”refers to the ability to increase or prolong, either in potency orduration, the effect of other therapeutic agents on a system. An“enhancing-effective amount,” as used herein, refers to an amountadequate to enhance the effect of another therapeutic agent in a desiredsystem.

The terms “co-administration” or the like, as used herein, are meant toencompass administration of the selected therapeutic agents to a singlepatient, and are intended to include treatment regimens in which theagents are administered by the same or different route of administrationor at the same or different time.

The term “carrier,” as used herein, refers to relatively nontoxicchemical compounds or agents that facilitate the incorporation of acompound into cells or tissues.

The term “diluent” refers to chemical compounds that are used to dilutethe compound of interest prior to delivery. Diluents can also be used tostabilize compounds because they can provide a more stable environment.Salts dissolved in buffered solutions (which also can provide pH controlor maintenance) are utilized as diluents in the art, including, but notlimited to a phosphate buffered saline solution.

A “metabolite” of a compound disclosed herein is a derivative of thatcompound that is formed when the compound is metabolized. The term“active metabolite” refers to a biologically active derivative of acompound that is formed when the compound is metabolized. The term“metabolized,” as used herein, refers to the sum of the processes(including, but not limited to, hydrolysis reactions and reactionscatalyzed by enzymes) by which a particular substance is changed by anorganism. Thus, enzymes may produce specific structural alterations to acompound. For example, cytochrome P450 catalyzes a variety of oxidativeand reductive reactions while uridine diphosphate glucuronyltransferasescatalyze the transfer of an activated glucuronic-acid molecule toaromatic alcohols, aliphatic alcohols, carboxylic acids, amines and freesulphydryl groups. Further information on metabolism may be obtainedfrom The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill(19%). Metabolites of the compounds disclosed herein can be identifiedeither by administration of compounds to a host and analysis of tissuesamples from the host, or by incubation of compounds with hepatic cellsin vitro and analysis of the resulting compounds.

“Bioavailability” refers to the percentage of the weight of the compounddisclosed herein (e.g. a compound from the group of Compound A) that isdelivered into the general circulation of the animal or human beingstudied. The total exposure (AUC(0-∞)) of a drug when administeredintravenously is usually defined as 100% bioavailable (F %). “Oralbioavailability” refers to the extent to which a compound disclosedherein, is absorbed into the general circulation when the pharmaceuticalcomposition is taken orally as compared to intravenous injection.

“Blood plasma concentration” refers to the concentration of a compoundwith a structure from the group of Compound A, in the plasma componentof blood of a subject. It is understood that the plasma concentration ofcompounds described herein may vary significantly between subjects, dueto variability with respect to metabolism and/or possible interactionswith other therapeutic agents. In accordance with one embodimentdisclosed herein, the blood plasma concentration of the compoundsdisclosed herein may vary from subject to subject. Likewise, values suchas maximum plasma concentration (Cmax) or time to reach maximum plasmaconcentration (Tmax), or total area under the plasma concentration timecurve (AUC(0-∞)) may vary from subject to subject. Due to thisvariability, the amount necessary to constitute “a therapeuticallyeffective amount” of a compound may vary from subject to subject.

As used herein, “calcium homeostasis” refers to the maintenance of anoverall balance in intracellular calcium levels and movements, includingcalcium signaling, within a cell.

As used herein, “intracellular calcium” refers to calcium located in acell without specification of a particular cellular location. Incontrast, “cytosolic” or “cytoplasmic” with reference to calcium refersto calcium located in the cell cytoplasm.

As used herein, an effect on intracellular calcium is any alteration ofany aspect of intracellular calcium, including but not limited to, analteration in intracellular calcium levels and location and movement ofcalcium into, out of or within a cell or intracellular calcium store ororganelle. For example, an effect on intracellular calcium can be analteration of the properties, such as, for example, the kinetics,sensitivities, rate, amplitude, and electrophysiologicalcharacteristics, of calcium flux or movement that occurs in a cell orportion thereof. An effect on intracellular calcium can be an alterationin any intracellular calcium-modulating process, including,store-operated calcium entry, cytosolic calcium buffering, and calciumlevels in or movement of calcium into, out of or within an intracellularcalcium store. Any of these aspects can be assessed in a variety of waysincluding, but not limited to, evaluation of calcium or other ion(particularly cation) levels, movement of calcium or other ion(particularly cation), fluctuations in calcium or other ion(particularly cation) levels, kinetics of calcium or other ion(particularly cation) fluxes and/or transport of calcium or other ion(particularly cation) through a membrane. An alteration can be any suchchange that is statistically significant. Thus, for example ifintracellular calcium in a test cell and a control cell is said todiffer, such difference can be a statistically significant difference.

As used herein, “involved in” with respect to the relationship between aprotein and an aspect of intracellular calcium or intracellular calciumregulation means that when expression or activity of the protein in acell is reduced, altered or eliminated, there is a concomitant orassociated reduction, alteration or elimination of one or more aspectsof intracellular calcium or intracellular calcium regulation. Such analteration or reduction in expression or activity can occur by virtue ofan alteration of expression of a gene encoding the protein or byaltering the levels of the protein. A protein involved in an aspect ofintracellular calcium, such as, for example, store-operated calciumentry, thus, can be one that provides for or participates in an aspectof intracellular calcium or intracellular calcium regulation. Forexample, a protein that provides for store-operated calcium entry can bea STIM protein and/or an Orai protein.

As used herein, a protein that is a component of a calcium channel is aprotein that participates in multi-protein complex that forms thechannel.

As used herein, “basal” or “resting” with reference to cytosolic calciumlevels refers to the concentration of calcium in the cytoplasm of acell, such as, for example, an unstimulated cell, that has not beensubjected to a condition that results in movement of calcium into or outof the cell or within the cell. The basal or resting cytosolic calciumlevel can be the concentration of free calcium (i.e., calcium that isnot bound to a cellular calcium-binding substance) in the cytoplasm of acell, such as, for example, an unstimulated cell, that has not beensubjected to a condition that results in movement of calcium into or outof the cell.

As used herein, “movement” with respect to ions, including cations,e.g., calcium, refers to movement or relocation, such as for exampleflux, of ions into, out of, or within a cell. Thus, movement of ions canbe, for example, movement of ions from the extracellular medium into acell, from within a cell to the extracellular medium, from within anintracellular organelle or storage site to the cytosol, from the cytosolinto an intracellular organelle or storage site, from one intracellularorganelle or storage site to another intracellular organelle or storagesite, from the extracellular medium into an intracellular organelle orstorage site, from an intracellular organelle or storage site to theextracellular medium and from one location to another within the cellcytoplasm.

As used herein, “cation entry” or “calcium entry” into a cell refers toentry of cations, such as calcium, into an intracellular location, suchas the cytoplasm of a cell or into the lumen of an intracellularorganelle or storage site. Thus, cation entry can be, for example, themovement of cations into the cell cytoplasm from the extracellularmedium or from an intracellular organelle or storage site, or themovement of cations into an intracellular organelle or storage site fromthe cytoplasm or extracellular medium. Movement of calcium into thecytoplasm from an intracellular organelle or storage site is alsoreferred to as “calcium release” from the organelle or storage site.

As used herein, “protein that modulates intracellular calcium” refers toany cellular protein that is involved in regulating, controlling and/oraltering intracellular calcium. For example, such a protein can beinvolved in altering or adjusting intracellular calcium in a number ofways, including, but not limited to, through the maintenance of restingor basal cytoplasmic calcium levels, or through involvement in acellular response to a signal that is transmitted in a cell through amechanism that includes a deviation in intracellular calcium fromresting or basal states. In the context of a “protein that modulatesintracellular calcium,” a “cellular” protein is one that is associatedwith a cell, such as, for example, a cytoplasmic protein, a plasmamembrane-associated protein or an intracellular membrane protein.Proteins that modulate intracellular calcium include, but are notlimited to, ion transport proteins, calcium-binding proteins andregulatory proteins that regulate ion transport proteins.

As used herein, the term “ameliorate” means to reduce, prevent,alleviate, and/or lessen the impact of a disease, symptom or condition,to bring about improvement in a disease or condition or at least apartial relief of symptoms associated with a disease or condition up toand including complete reduction such that said impact is zero oreffectively zero.

As used herein, “cell response” refers to any cellular response thatresults from ion movement into or out of a cell or within a cell. Thecell response may be associated with any cellular activity that isdependent, at least in part, on ions such as, for example, calcium. Suchactivities may include, for example, cellular activation, geneexpression, endocytosis, exocytosis, cellular trafficking and apoptoticcell death.

As used herein. “immune cells” include cells of the immune system andcells that perform a function or activity in an immune response, suchas, but not limited to, T-cells, B-cells, lymphocytes, macrophages,dendritic cells, neutrophils, eosinophils, basophils, mast cells, plasmacells, white blood cells, antigen presenting cells and natural killercells.

As used herein, “cytokine” refers to small soluble proteins secreted bycells that can alter the behavior or properties of the secreting cell oranother cell. Cytokines bind to cytokine receptors and trigger abehavior or property within the cell, for example, cell proliferation,death or differentiation. Exemplary cytokines include, but are notlimited to, interleukins (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18,IL-la, IL-10, and IL-1 RA), granulocyte colony stimulating factor(G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF),oncostatin M, erythropoietin, leukemia inhibitory factor (LIF),interferons, B7.1 (also known as CD80), B7.2 (also known as B70, CD86),TNF family members (TNF-α, TNF-β, LT-β, CD40 ligand, Fas ligand, CD27ligand, CD30 ligand, 4-1BBL, Trail), and MIF.

“Store operated calcium entry” or “SOCE” refers to the mechanism bywhich release of calcium ions from intracellular stores is coordinatedwith ion influx across the plasma membrane.

“Selective inhibitor of SOC channel activity” means that the inhibitoris selective for SOC channels and does not substantially affect theactivity of other types of ion channels.

“Selective inhibitor of CRAC channel activity” means that the inhibitoris selective for CRAC channels and does not substantially affect theactivity of other types of ion channels and/or other SOC channels.

As used herein, the term ‘calcium’ may be used to refer to the elementor to the divalent cation Ca²⁺.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES Example 1: CRAC Channel Inhibition by GSK-7975A Blocks Necrosisin Mouse and Human Pancreatic Acinar Cells

Mouse Pancreatic Acinar Cells (PACs) were extracted and incubated witheither a carrier (control) or with natural bile acid TLCS(taurolithocholic acid 3-sulfate) in the absence or presence ofGSK-7975A. Cells were contacted with propidium iodide to assay for cellnecrosis. TLCS treatment to individual cells in vitro mimics the effectof a gall stone or other blockage in pancreatic secretion in vivo.

As indicated in FIG. 1A, TLCS induced necrosis in about 45% of cellsduring the time period of the experiment. Addition of GSK-7975A at 10 μMreduced this percent necrosis by half, to about 23%. The asteriskindicates a statistically significant change. Cells not treated withTLCS demonstrated a necrosis of about 10%. This result demonstrates thatGSK-7975A reduces the necrosis-inducing effect of TLCS on mouse PACs.

Human PACs were extracted and incubated with either a carrier (control)or with natural bile acid TLCS in the absence or presence of GSK-7975A.Cells were contacted with propidium iodide to assay for cell necrosis.TLCS treatment to individual cells in vitro mimics the effect of a gallstone or other blockage in pancreatic secretion in vivo.

As indicated in FIG. 1B, TLCS induced necrosis in about 45% of cellsduring the time period of the experiment. Addition of GSK-7975A at 10 μMreduced this percent necrosis to about 30%. The asterisk indicates astatistically significant change. Cells not treated with TLCSdemonstrated necrosis at about 23%. This result demonstrates thatGSK-7975A reduces the necrosis-inducing effect of TLCS on human PACs.

Example 2: CRAC Channel Inhibitor (GSK-7975A) Blocks HistopathologicalChanges in Mouse Models of AP

Mouse acute pancreatitis models were used to evaluate the effect of aCRAC inhibitor on pancreas histopathological progression. Caerulein isused to hyper-stimulate CCK receptors in the normal Calcium signalingpathway in the mouse pancreas. TLCS is used to induce acute pancreatitisthrough simulation of an excess of bile acid, as would be experienced ina gallstone induced acute pancreatitis. Fatty acid ethyl esters (FAEE)are used to simulate alcohol-induced acute pancreatitis. Mice weretreated with the acute pancreatitis agent (Caerulin, FIG. 2A, TLCS. FIG.2B or FAEE, FIG. 2C) alone or with the CRAC inhibitor GSK-7975A ateither 10× or 40× the IC₅₀ of the CRAC inhibitor.

It is observed that GSK-7975A substantially reduces the totalhistopathology score of treated mice relative to mice treated with theagent in the absence of the CRAC inhibitor. This effect wasstatistically significant and substantially more pronounced at 40× IC₅₀that ant 10×IC₅₀, but was observed at the lower concentration as well.The asterisk indicates a statistically significant change. GSK-7975A atdoses that achieve tissue levels 10- or 40-fold above its in vitro IC₅₀value produces significant reductions in pancreatic histopathology.

The results presented in FIGS. 2A, 2B and 2C indicate that a CRACinhibitor can ameliorate the histopathological symptoms of acutepancreatitis. This effect is observed independent of the inducing agentor the type of acute pancreatitis being modeled.

Example 3: Compound I and GSK-7975A Inhibit CRAC Channels

Compound I and GSK-7975A were assayed for their inhibitory effect onCRAC channels. Channels comprising Orai1/STIM1 and Orai2/STIM1 wereassayed. As presented in FIG. 3A, it was determined that Compound Iinhibited Orai1/STIM1 channels with a mean IC₅₀ of 119 nM, andOrai2/STIM1 channels with a mean IC₅₀ of 895 nM. As presented in FIG.3B, it was determined that GSK-7975A inhibited Orai1/STIM1 cannels witha mean IC₅₀ of 398 nM, and Orai2/STIM1 channels with a mean IC₅₀ of 1453nM. Compound I is about 4-fold more potent on Orai1-type CRAC channelscompared to GSK-7975A. Both compounds are more potent on Orai1 thanOrai2-type CRAC channels.

These results indicate that the effect observed for GSK-7975A on Calciumsignaling is generalizeable to CRAC inhibitors, and that CRAC inhibitorsother than GSK-7975A may outperform GSK-7975A on some parameters.

Example 4: Compound I Blocks Store-Operated Ca²⁺ Entry (SOCE) in MousePancreatic Acinar Cells

Mouse PACs were isolated and assayed for the effect of CRAC inhibitorson calcium re-uptake into the ER. Cells were treated with cyclopiazonicacid (CPA) alone (FIG. 4A) or in combination with the CRAC inhibitorCompound I (FIG. 4B) to activate CRAC channels to release Ca²⁺. At 15minutes following Calcium release, cells were provided with an excess ofCalcium and monitored for Calcium uptake into the ER. It is observedthat the CRAC inhibitor-treated cells do not demonstrate reuptake ofCalcium.

This result indicates that CRAC inhibitors may block the cell's abilityto reload its ER with Calcium for successive rounds of signaling.

Example 5: Compound I and GSK-7975A Block SOCE in Mouse PancreaticAcinar Cells in a Dose-Dependent Manner

Mouse PACs were treated with CRAC inhibitors Compound I (FIG. 5A) orGSK-7975A (FIG. 5B) and monitored for their rate of Calcium uptake. BothCRAC inhibitors reduced the rate of store-operated Calcium entry intothe ER to 50% of control levels upon treatment with 700 nM of inhibitor.Compound I blocks 100% of reuptake at 10 mM.

This example demonstrates that multiple CRAC inhibitors each act toinhibit SOCE in mammalian PACs.

Example 6: Compound I Blocks CCK-Induced Ca²⁺ Entry in Mouse PancreaticAcinar Cells

Mouse PACs were monitored for their Calcium uptake upon treatment withCCK at 10 nM. Cells were treated with CCK and then provided with 1.8 mMCalcium and Calcium reuptake was monitored. It was observed that cellspre-treated with CRAC inhibitors (FIG. 6B) demonstrated substantiallyreduced Calcium re-uptake compared to untreated cells (FIG. 6A).Compound I pretreatment reduced Calcium re-uptake to near 0% of thecontrol. GSK-7975A reduced calcium reuptake to about 30% of the control.

These results indicate than CRAC inhibitors may be efficacious inreducing overactive Calcium signaling in PACs.

Example 7: Compound I Inhibits Multiple Cytokines

Compound I was tested for its inhibitory effect on a number ofcytokines. Cytokines INF-gamma, IL-4, and IL-4 receptors of which areexpressed on Acinar cells, cytokines IL-1beta, IL-6, IL-10 andTNF-alpha, expressed in Acinar cells, and IL-2 and IL-7, cytokinesimportant in T-cell function, were tested for an inhibitory effect ofthe CRAC inhibitor Compound I. T cells in bulk human PBMCs werestimulated with plate-bound anti-CD3/anti-CD28 in buffer+10% serum for48 hrs. Released cytokines were measured by Luminex at Millipore. Theresults are presented in FIG. 7. In human PBMCs, Compound I potentlyinhibits release of multiple cytokines which play important roles in Tcells.

These cytokine data, together with the PAC data, support the conclusionthat Compound I has a dual effect in acute pancreatitis, inhibiting bothimmune cell and pancreatic acinar cell signaling pathways and death.

Example 8: Compound I Shows Robust Efficacy in a Mouse Calcinurin Modelof Acute Pancreatitis

Mice were treated with a CRAC inhibitor or a vehicle prophylactically,and then challenged with CCK to trigger acute pancreatitis. Compound I,administered i.p. prophylactically, produced significant anddose-dependent reductions in caerulein-induced pathology in the mousepancreas (C₅7B6 mice). This effect was significantly below the positivecontrol CsA at 5 mg/kg, and increased in a dose-dependent manner. SeeFIG. 8A. The treatment demonstrated a dose-proportional increase ofCompound I levels in the pancreas after i.p. injection (see FIG. 8B),which corresponded to the positive results in FIG. 8A, above.

These results indicate that CRAC administration, prior to orconcurrently with a second drug suspected of triggering acutepancreatitis or increasing the risk of acute pancreatitis, mayprophylactically protect against or reduce the risk of acutepancreatitis.

Example 9: Compound I Lowers Serum Amylase and Serum Lipase Levels inMouse Caerulein Model of Acute Pancreatitis

Mice were either untreated (Normal), treated with CCK only (vehicle), ortreated with CCK in combination with CsA or a CRAC inhibitor at theindicated dose. Serum amylase activity (FIG. 9A) and Serum Lipaseactivity (FIG. 9B) (IU/L) were measured.

The CRAC inhibitor Compound I performed as well as or better than thepositive control CsA at maximal concentrations of 20 mg/kg. Compound Iproduced significant and dose-dependent reductions in caerulein-inducedserum amylase and serum lipase activities

Example 10: Compound I Reduces Pancreatic Pathology in Therapeutic MouseCaerulein Model

Seven hourly i.p. injections of caerulein were given to inducepancreatitis, with animals sacrificed 8 hr after the 1st injection.Compound I was administered i.p. either 30 min before the 1st caeruleininjection (prophylactic), or after the 3rd injection (therapeutic). CsAwas administered p.o. on the same schedule as Compound I. The resultsare depicted in FIG. 10.

Histopathology scores were measured in light of observation of Acinarcell degeneration, coagulation necrosis, and inflammation and edemameasurements. Compound I produced a significant 35% reduction incaerulein-induced pathology in the pancreas when administered after the3rd caerulein injection, consistent with efficacy in a therapeutic mode.Prophylactic treatment with Compound I also reduced pathology by 26%.

The results indicate that CRAC inhibitors such as Compound I have asubstantial effect on pancreas histopathology scores when administeredprophylactically or therapeutically.

Example 11: Compound I Blocks TLCS-Induced Ca²⁺ Entry in MousePancreatic Acinar Cells

Mouse PACs were treated with 500 μM TLCS and either 0.1 μM or 3 μM ofthe CRAC inhibitor Compound I. Cytosolic Calcium levels (measured asF₃45/F₃80 ratios) were measured for each treatment. TLCS releases Ca²⁺from intracellular stores (not shown), initiating SOCE. In thisexperiment, Compound I at 1 or 3 μM completely blocked TLCS-induced Ca²⁺entry. The results are depicted in FIG. 11.

This result indicates that CRAC inhibitors may be effective atpositively influencing Calcium signaling in Gall stone related acutepancreatitis in mammals.

Example 12: Compound I and Other CCIs Inhibit TLCS-Induced AmylaseRelease in Mouse Pancreatic Acinar Cells

Amylase release from PACs has an ER calcium-dependent and aCRAC/cytosolic calcium-dependent component. The calcium dependentcomponent is blocked by introduction of the divalent cation chelatingagent EGTA. Mouse Acinar cells were treated with TLCS and eithervehicle, EGTA, or a CRAC inhibitor such as Compound I, GSK-7975A orCompound II and monitored for Amylase release. It is observed that CRACinhibitors mimicked EGTA in their effect on Amylase release. See FIG.12.

This result indicates that CRAC inhibitors block the re-uptake ofCalcium into the ER following TLCS-induced calcium release, therebymimicking the effect of EGTA on the inhibition of Amylase release.

Example 13: Compound I Inhibits TLCS-Induced Necrosis in MousePancreatic Acinar Cells

Mouse PACs were treated with either DMSO, DMSO plus 500 μM TLCS, or DMSOplus 500 μM TLCS plus 1 μM of the CRAC inhibitor Compound I. Cellnecrosis was measured as % P uptake. The CRAC inhibitor Compound Iinhibits TLCS-induced necrosis in mouse PACs.

These data, together with the Ca²⁺ and amylase data, indicate thatCompound I is efficacious in the TLCS model of AP.

Example 14: Phase II Clinical Trial of the Safety and Efficacy ofCompounds Compound I, GSK-7975A, and Compound II in Patients with AcutePancreatitis

The purpose of this phase II trial is to investigate the safety,tolerability, PK, PD, and efficacy of single and repeat intravenousinfusions of a Calcium signaling inhibitor such as Compound I,GSK-7975A, and Compound II, or compounds selected from the group ofCompound A, in patients with acute pancreatitis and accompanying SIRS.

Patients:

The study will enroll 30 patients at high risk of developing moderate orsevere pancreatitis, as assessed by a SIRS score of 2 or more at studyentry.

Criteria:

Inclusion Criteria:

-   -   All subjects must use acceptable contraception to ensure that no        pregnancies occur during the course of the study and for at        least 12 weeks after dosing for males and for 32 weeks after        dosing for females;    -   Body mass index within the range 18.5-35 kg/m² inclusive, in        addition to a weight range of 55-95 kg;    -   The subject must be capable of giving informed consent and can        comply with the study requirements and timetable;    -   Male and female subjects age 18 or higher are eligible.    -   Subjects must be experiencing a first in a lifetime episode of        acute pancreatitis.    -   Diagnosis of acute pancreatitis must be based on 2 of the        following 3 criteria: (1) typical upper abdominal pain; (2)        elevation of serum amylase and/or lipase at least 3 times the        upper limit of normal; (3) contrast-material enhanced CT scan or        abdominal sonogram demonstrating changes of acute pancreatitis.    -   Subjects must demonstrate a history supporting alcoholic,        hypertriglyceridemic or biliary etiology of the current        pancreatitis episode (for biliary pancreatitis, a sonogram must        exclude a stone obstruction at the time of study screening).    -   Subjects must demonstrate a BISAP score of 3 or higher    -   Study treatment initiation is possible within 48 h of symptom        onset

Exclusion Criteria:

-   -   High likelihood for an invasive intra-biliary tract intervention        (e.g. ERCP) in the coming week.    -   Recurrent episode of pancreatitis.    -   CT evidence of pancreatic necrosis at study entry.    -   Severe chronic renal failure (Modification of Diet in Renal        Disease formula 30 mL/min or dependency on renal dialysis).    -   Class II or greater New York Heart Association heart failure.    -   Oxygen-dependent chronic obstructive pulmonary disease (COPD).    -   Cirrhosis of the liver.    -   Severe anemia (hemoglobin less than 8 g/dL).    -   Hematocrit below 35% or above 45% at study entry (fluids may be        administered to correct the hematocrit before randomization as        long as study treatment starts within 48 hours of symptoms        onset).    -   Serum alanine aminotransferase above 250 IU/L at study entry.    -   Clinical suspicion of ascending cholangitis at study entry.    -   Active gastrointestinal bleeding.    -   Current malignancy not in remission (other than basal cell        carcinoma of skin).    -   Altered mental status.    -   Current breastfeeding or pregnancy.    -   Female of childbearing potential (less than 2 years        postmenopausal or not surgically sterilized) who is not willing        to use adequate and effective birth control measures    -   Known hypersensitivity to any component of the investigational        product.    -   Dependent relationship with the investigator or the sponsor.    -   Participation in an investigational drug study during this        clinical trial or within 30 days prior to start of this clinical        trial.

Study Design:

The study is a randomized, double blind, placebo-controlled,multi-center, multi-national, parallel-arm study comparing a placebogroup to a CRAC inhibitor group treated intravenously with a CRACinhibitor twice daily for up to 7 consecutive days.

The study will enroll 45 patients at high risk of developing moderate orsevere pancreatitis, as assessed by a SIRS score of 2 or more at studyentry.

The primary study endpoint is the effect of a CRAC inhibitor on systemicinflammation in acute pancreatitis as reflected by a change in SIRSscore or C-reactive protein (CRP) plasma levels.

Clinical trial material administration will begin within 24 hours ofacute pancreatitis symptoms onset, or 18 hours of hospital admittance.Subjects will be randomized at a ratio of 1:1 to receive either a CRACinhibitor at one of two doses, or placebo. The serum levels of a CRACinhibitor at the end of each 2-hour infusion will also be monitored.

The study duration per individual subject will be 14 days, consisting ofa screening evaluation followed by an up to 7 day double blind treatmentperiod, which will be part of an in-hospital observation period of atleast 7 days, and a follow-up final visit on Day 14.

Primary Outcome Measures:

-   -   C-reactive protein serum concentration.    -   Change in SIRS at 48 hours    -   Blood amylase and lipase level.

Secondary Outcome Measures, CRAC Inhibitor Vs. Placebo:

-   -   Safety of a CRAC inhibitor in this population of patients        (through routine safety laboratory tests,    -   Physical examination and vital signs monitoring, ECG and adverse        event reporting),    -   A CRAC inhibitor's effects on other plasma inflammatory markers        (interleukin-6, matrix metalloproteinase 9, tumor necrosis        factor alpha, etc.)    -   CRAC inhibitor effects on the clinical course of pancreatitis        (based on changes in clinical rating scales such as the Bedside        Index for Severity in Acute Pancreatitis (BISAP), Systemic        Inflammatory Response Syndrome (SIRS) and Acute Physiology And        Chronic Health Evaluation II (APACHE II) scores, and on contrast        material-enhanced abdominal Computerized Tomography (CT),    -   Progression in the Sequential Organ Failure Assessment (SOFA)        Score    -   Progression in the Multiple Organ Dysfunction Score (MODS)    -   Progression in the Systemic Inflammatory Response Syndrome    -   Progression in the inflammatory and anti-inflammatory mediators        (IL-1RA, IL-10, IL-6. IL-18, TNF-α, ICAM-1, IL-10 etc.).    -   Escalation of care to high dependency or intensive care unit and        length of hospital stay.

Example 15: Compound I Inhibits Junin Virus Budding in Infected VeroE6Cells

VeroE6 cells infected with live attenuated Candid-1 JUNV are treatedwith either DMSO, DMSO plus 500 μM TLCS, DMSO plus 500 μM TLCS plus 1 μMof the CRAC inhibitor Compound I, DMSO plus 500 μM TLCS plus 10 μMCompound I, DMSO plus 500 μM TLCS plus 25 μM Compound I, or DMSO plus500 μM TLCS plus 50 μM Compound I. Infectious virions produced fromthese cells are quantified in a focus-forming assay. Enumeration of JUNVfoci reveals a statistically significant, dose-dependent reduction inJUNV virus production following treatment with Compound I.

These data, together with the Ca²⁺ data, indicate that Compound I isefficacious in inhibiting viral budding of the JUNV virus.

Example 16: Compound I Differentially Inhibits Store-Operated Ca²⁺Entry-Dependent Th1 Th2, and Th17 Differentiation

Naive and stimulated murine T cells cultured under Th1-, Th2-, andTh17-polarizing conditions are treated with either DMSO, DMSO plus 500μM TLCS, or DMSO plus 500 μM TLCS plus 1 μM of the CRAC inhibitorCompound I. Store-operated Ca²⁺ entry (SOCE) is quantified in eachtreatment. Compound I blocks SOCE stronger in differentiated Th17 cellsthan in naive, Th1, or Th2 cells. In addition, IL-17A production by Th17cells is more severely affected when compared with IFN-γ and IL-4production by Th1 and Th2 cells, respectively, in the presence ofCompound I.

These data, together with the Ca²⁺ and additional transcription factor(i.e., IL-17A, RORα, and RORγt) expression data, indicate that CompoundI is efficacious in inhibiting Th17 differentiation.

1. A compound having the chemical nameN-(5-(6-chloro-2,2-difluorobenzo[d][1,3]dioxol-5-yl)pyrazin-2-yl)-2-fluoro-6-methylbenzamideor a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising the compound of claim 1 and a pharmaceuticallyacceptable excipient thereof.